A simple way to batch propagate direct somatic embryos and kanamycin resistance of Portulaca amilis (Portulacaceae)

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A successful somatic embryogenesis protocol requires optimisation on numerous factors such as growth regulators, tissue type, light, temperature, survival rate and can be species specific. Due to lack of callus phase, direct somatic embryogenesis is usually more efficient but can be recalcitrant to induce. In this study, I established an efficient, simple way to induce, regenerate and acclimatise direct somatic embryos from various young shoot tissues of three-week-old seedlings in Portulaca amilis (Portulacaceae) with high survival rate. The regenerated plantlets can be maintained in sterile environment and set seeds in vitro or acclimatised and set seeds in non-sterile environment, flexible for different purposes. The species has great potential as a model species for biochemical, physiological, medical, genetical and morphological studies. The only external hormone is 1mg/L 6-benzylaminopurine and it only takes three to four months from preparing explants from sterile seeds to mature regenerated plants setting seeds in non-sterile glasshouses. I also found out the threshold kanamycin concentration in both tissue (50mg/L) and seeds (100mg/L) for P. amilis . Both direct somatic embryogenesis and kanamycin resistance will be informative for future genetic studies involving transformation. Portulaca amilis direct somatic embryogenesis kanamycin resistance tissue culture Portulacaceae Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key message Direct somatic embryogenesis occurs in young shoot tissues of with 1mg/L BAP on full MS medium. Kanamycin resistance threshold is 50mg/L for shoot tissues and 100mg/L for seeds. 1. Introduction Plant somatic embryogenesis has many critical applications in science, agriculture and industry, such as plant breeding, improving crop yield, medicine production, gene transformation and testing gene functions (Raemakers et al., 1995 ; Birch, 1997 ; Hansen and Wright, 1999 ; Newell, 2000 ; Kamle et al., 2011 ; Sedaghati et al., 2019 ). In the past few decades, people have reported at least 200 species in seed plants that are capable of direct to indirect somatic embryogenesis, varying degrees on a spectrum (Williams and Maheswaran, 1986 ; Wann, 1988 ; Carman, 1990 ; Raemakers et al., 1995 ). The key distinction between direct and indirect somatic embryogenesis (i.e. two extremes on the direct-indirect spectrum) is whether the tissue undergoes the callus phase before producing embryos (Sharp et al., 1980 ; Evans et al., 1981 ). And due to the callus phase and tissue re-differentiation, it generally takes much longer for indirect somatic embryogenesis within a species (vary from weeks to years, e.g. Davidonis and Hamilton, 1983 ; Sedaghati et al., 2019 ). Somatic embryogenesis can sometimes be difficult to induce and optimise, as there are numerous factors involved. The main factors involved are tissue types, species (include specific genotype/varieties) and growth hormones (e.g. Fillatti et al., 1987 ; Lowe et al., 2018 ; Sun et al., 2018 ; Sedaghati et al., 2019 ). For example, the optimised protocol for cotton ( Gossypium ) and tomato ( Solanum lycopersicum ) are both via indirect somatic embryogenesis using hypocotyl or cotyledon (Fillatti et al., 1987 ; Godishala et al., 2011 ; Juturu et al., 2015 ). In Portulaca oleracea , leaves can produce both direct and indirect somatic embryos while stems can only produce indirect ones under similar hormone combinations (Sedaghati et al., 2019 ). 2,4-dichlorophenoxyacetic acid (2,4-D), a synthetic auxin, has higher chance to induce abnormal somatic embryos in various crops or ornamental plants than other auxin or cytokinin derivatives (reviewed in Garcia et al., 2019 ). Apart from these factors, explant loss occurs in almost each step of somatic embryogenesis, from initial explant preparation (including sterilisation), embryogenesis to final acclimatisation and growth of somatic embryos to mature plants, so the final survival rate of mature plants can be low (Litz and Gray, 1995 ; Park et al., 1998 ). Therefore, optimising conditions for somatic embryogenesis can be time consuming and highly species dependent, yet a successful protocol will benefit numerous downstream research. In this study, I established an efficient, simple way to induce, regenerate and acclimatise direct somatic embryos from various young shoot tissues in Portulaca amilis (Portulacaceae) with high survival rate, along with its kanamycin resistance ability (Fig. 1 ). The regenerated plantlets can be maintained in sterile environment and set seeds in vitro or acclimatised and set seeds in non-sterile environment, flexible for different purposes. Members of Portulaca produce betalain, use C4-CAM photosynthesis, can be consumed as vegetables or medical herbs, and has unusual perianth evolutionary history (Kubitzki et al., 1993 ; Guralnick and Jackson, 2001 ; Brockington et al., 2009 ; Sedaghati et al. 2019 , 2021 ). They have great potential for biochemical, physiological, medical, genetical and morphological studies, and it is thus worth establishing a model species within this genus. P. amilis is potentially a better candidate model species than closely-related, well-studied species like P. oleracea , P. grandiflora and P. pilosa , despite all three having direct or indirect somatic embryogenesis/organogenesis protocols (Safdari and Kazemitabar, 2010 ; Sedaghati et al. 2019 , 2021 ; Chen et al., 2020 ; Xiong et al., 2021 ). First, apart from fast growth cycle and massive seed production shared by all Portulaca , P. amilis is a simple diploid instead of species complexes like P. oleracea and P. grandiflora , more suitable for gene knock-out (Danin et al., 1978 ; Mishiba and Mii, 2000 ; Walter et al., 2015 ). Second, the seeds are highly viable in long-term lab storage instead of low survival rate like P. pilosa (Kim and Carr, 1990 ), easier to maintain large lab populations. Third, P. amilis is the only Portulaca with a fully sequenced genome, available to refer and annotate gene functions (Gilman et al., 2022 ). Such advantages make P. amilis a better option for transgenic analyses on gene functions, especially genes related to betalain pathway and unusual perianth evolution, both of which are active research areas (Brockington et al., 2009 ; Sheehan et al., 2020 ). Moreover, transgenic analyses usually involve designing gene editing constructs (e.g. CRISPR) to silence, knockout or overexpress certain genes and searching for off-target effects, where somatic embryogenesis is required in selecting and regenerating successfully transformed individuals. As a result, the findings of this study on direct somatic embryogenesis and kanamycin resistance of P. amilis will benefit future experimental design in transformation, genetic analyses, mass propagation and medical applications of the species. 2. Materials and methods Since direct somatic embryogenesis is more efficient than the indirect one and can be highly tissue-dependent, I tested different shoot tissues’ ability on direct somatic embryogenesis in Portulaca amilis . Besides, as direct somatic embryogenesis is closely associated with plant transformation, which often use kanamycin as the selection agent in Agrobacterium- mediated transformation (e.g. Fillatti et al., 1987 ; Gatzak et al., 2002; Bagheri et al., 2010 ; Raut et al., 2016 ; Sedaghati et al. 2019 ), I tested both seed and different tissues’ ability on kanamycin resistance to prepare for future transformation experiments via tissue culture or seeds. The main experimental procedures were inspired by protocols of Sedaghati et al. ( 2019 , 2021 ) on tissue and seed transformation of P. oleracea , as P. oleracea is closely related to P. amilis . All P. amilis material used in this study originally came from United States, Department of Agriculture (PI 677126). 2.1-Explant preparation I sterilised ~ 1200 seeds with 70% ethanol for one minute, then with Milton Mini Tablet-sterilised water for 25 minutes (solution contains ~ 0.02% (m/m) sodium dichloroisocyanurate, 1 tablet in ~ 50ml sterile water), and finally washed the seeds three times with sterile water for 1 minute/time. ~200 seeds were prepared for tissue kanamycin test and direct somatic embryogenesis while the rest ~ 1000 were prepared for seed kanamycin test. For tissue kanamycin test and direct somatic embryogenesis, the sterile seeds were plated on full strength Murashige and Skoog ( 1962 ) medium (MS) with Gamborg B5 vitamins, supplemented with 3% (m/v) sucrose and 0.7% (m/v) bacto-agar. The pH of the medium was adjusted to 5.8 ± 0.05 by 0.5 mol/L KOH and 0.5 mol/L HCl before autoclaving. I germinated and grew these seeds in a growth chamber (Panasonic MLR-352-PE Climate Chamber), and set the conditions to 25°C, with 16h light/8h dark photoperiod and light intensity of ~ 5300 Lux. When the seedlings reached three weeks old, I cut and separated each seedling into three explant types: hypocotyl, true leaves and shoot apical meristem (Fig. 1 c-d). I kept ~ 30 seedlings growing for another three weeks and collected young leaves (~ 5-7mm in length) from these six-week-old plants as the fourth explant type. I used all four explant types in the kanamycin resistance test and direct somatic embryogenesis. For seed kanamycin test, the sterile seeds were soaked in a 10ml sterile conical flask filled with sterile liquid medium (full strength MS with Gamborg’s B5 vitamins, supplemented with 3% (m/v) sucrose, pH = 5.8 ± 0.05). I sealed the flask and shook it gently (120rpm) in darkness at room temperature for 24 ~ 36h before the seeds were ready for plating. The soaking mimics the environment before and during seed transformation. 2.2-Setups for kanamycin (Km) resistance test 2.2.1-Tissue kanamycin test To find out the threshold kanamycin concentration ([Km]) that inhibits various tissue growth, I set up a kanamycin concentration gradient ([Km] = 0 (control), 50, 100, 150, 200, 250mg/L) on full strength MS medium supplemented with 3% (m/v) sucrose, 0.7% (m/v) bacto-agar, 1mg/L (hypocotyl and shoot apical meristem) or 1.5mg/L (leaf) of 6-benzylaminopurine (BAP), pH = 5.8 ± 0.05. For each explant type under a certain [Km], I had two plates as biological replicates and each plate had 14 explants. Therefore, there were 14 explants/plate × 2 plates/(tissue type × concentration group) × 4 tissue types × 6 concentration groups = 672 explants. The explants were cultured on the corresponding medium in the growth chamber at 25°C, first in complete darkness for three weeks (dark period), then in 16h light/8h dark photoperiod with light intensity of ~ 5300 Lux for another three weeks (light period). I resupplied the explants with fresh media once every three weeks. By the end of the light period (the sixth week from putting freshly-cut explants on plates), I recorded the number of survived explants on each plate, described their colour (i.e. bleached/unbleached), and the number of explants that produced callus and direct somatic embryos. I used generalised linear modelling and analysis of covariance for binominal data in R (version 4.2.1) to look for the threshold kanamycin concentration that inhibits explant growth. 2.2.2-Seed kanamycin test To find out the threshold kanamycin concentration ([Km]) that inhibits seed growth, I set up a kanamycin concentration gradient ([Km] = 0 (control), 50, 100, 150, 200, 250mg/L) on full strength MS medium with Gamborg’s B5 vitamins, supplemented with 3% (m/v) sucrose, 0.7% (m/v) bacto-agar, pH = 5.8 ± 0.05. For each Km concentration, I had three plates and I plated 50 seeds/plate. Therefore, I had 3 plates/concentration group × 6 concentration groups = 18 plates, and 18 plates × 50 seed/plate = 900 seeds in total. I grew the seeds in the growth chamber under 16h light/8h dark photoperiod (light intensity of ~ 5300 Lux) at 25°C for three weeks. By the end of the third week, I recorded several parameters for each plate, including the number of seeds that germinated, produced true leaves, produced side roots, fell down and were bleached after kanamycin treatment. I also randomly chose 10 germinated seeds from each plate and estimated the relative size of their cotyledons and first true leaves by measuring the length and width of each leaf and calculating the product of these two parameters. I analysed these data using either generalised linear modelling for binomial data, or one-way (nested) ANOVA under either parametric or non-parametric assumptions in R (version 4.2.1). 2.3-Direct somatic embryogenesis and acclimatisation for somatic embryos Since the control group of tissue kanamycin test ([Km]=0mg/L) shared the same growth condition as direct somatic embryogenesis, after the kanamycin resistance test, I used all surviving explants in the control group to test the ability of various tissues to produce direct somatic embryos. To induce direct somatic embryos, I continued to keep these explants under the same temperature and light conditions as the ‘light period’ in the kanamycin resistance test, and resupplied them with fresh control group media once every three weeks. Once the diameter of the embryos reached ≥5mm and became mini plantlets, I cut the regenerated plantlets out of the original explants and put them in rooting medium without BAP (full strength MS medium supplemented with 3% (m/v) sucrose, 0.7% (m/v) bacto-agar, pH = 5.8 ± 0.05) under the same temperature and light conditions. When the roots of the regenerated plantlets reached about 1 ~ 2cm, I gradually acclimatised the plantlets to a non-sterile environment with fluctuating temperature. I took the rooted plantlets out of the media, washed away the media with sterile water and grew them in 2ml centrifuge tubes filled with sterile water for three to four days (Fig. 2 a). All centrifuge tubes were kept in a clean 1000µl pipette tip box with the box lid closed in the growth chamber (Fig. 2 a). Then I moved these rooted plantlets from sterile water to sterile soil in 3×3×4cm small pots (M3 soil:sand:perlite = 2:2:1) (Fig. 2 b). I also kept the pots in clean 1000µl pipette tip boxes in the growth chamber and gradually opened the box lid to acclimatise the rooted plantlets to a non-sterile environment (Fig. 2 b). These rooted plantlets were watered once a day or once every two days based on soil moisture. When the rooted plantlets had acclimatised in the soil for more than a week and reached about 3-5cm in height, I moved them to the glasshouse (16h light/8h dark photoperiod, light intensity ~ 5300 Lux but varied a bit based on weather, temperature 18 ~ 25°C) and transplanted them to larger pots in the same soil (non-sterile). These plants were watered by Cambridge University Botanic Garden staffs on a regular basis. On the 10th week from putting freshly cut explants on plates (i.e. four weeks after kanamycin resistance test/seven weeks into light period), I recorded the type and the number of explants that directly produced somatic embryos, and the approximate number of embryos produced by a single explant. By the end of the whole experiment (i.e. 16 weeks after the kanamycin resistance test), I recorded the total number of mini plantlets that were cut out from each type of explant, the source (i.e. which explant type the plantlet originated from) and number of plantlets that successfully rooted in the rooting medium, and the source and number of regenerated plantlets that survived the whole acclimatising process. For this set of data, I either used generalised linear models for binominal data in R (version 4.2.1) or directly summarised the data into tables to look for which types of tissue are better at surviving and producing direct somatic embryos under control conditions ([Km]=0mg/L). 3. Results 3.1-Kanamycin (Km) resistance test 3.1.1-Tissue Km threshold As soon as kanamycin (Km) was present at 50mg/L, all tissue types were more or less negatively affected (Fig. 3 ). The proportion of surviving true leaves from both 3-week-old seedlings and 6-week-old plants decreased quickly with increased Km concentration ([Km]), while the survival of hypocotyl and shoot apical meristem (SAM) were less negatively affected (Fig. 3 a; analysis of covariance (ANCOVA) for binomial data; p < 0.001 for both [Km]—χ 2 = 16.88 and tissue type— χ 2 = 557.98, df = 3). At [Km]=0mg/L, both types of true leaves were significantly better at producing callus, while hypocotyl and SAM were significantly better at producing direct somatic embryos, and these regeneration activities soon predicted to decrease to 0 when [Km]≥50mg/L (Fig. 3 b,c; ANCOVA for binomial data; for callus, p < 0.001 for both [Km]—χ 2 = 123.29 and tissue type—χ 2 = 83.45, df = 3; for direct somatic embryos, p < 0.001 for [Km]—χ 2 = 29.16, p = 0.0064 for tissue type—χ 2 = 12.30, df = 3). Moreover, the proportion of bleached tissues quickly increased from 0% to 100% for all tissue types when [Km]≥50mg/L (Fig. 3 d; ANCOVA for binomial data; p < 0.001 for [Km] and non-significant for tissue type). Based on these results, the threshold kanamycin concentration is 50mg/L for various tissue type from seedlings or young plants. 3.1.1-Seed Km threshold Overall, kanamycin (Km) had strong negative effects on all recorded parameters except seed germination (Fig. 4 ). Seed germination rate slowly increased with increasing Km concentration ([Km]) (Fig. 4 a, light green line; p < 0.001, logistic generalised linear model for binomial data, χ 2 = 26.71). On the other hand, the proportion of germinated seeds that produced true leaves quickly decreased with increasing [Km] (Fig. 4 a, dark green line; p < 0.001, logistic generalised linear model for binomial data, χ 2 = 538.39)—almost all germinated seeds produced true leaves when there was no Km, while only ~ 40% of germinated seeds produced true leaves under [Km]=50mg/L and their true leaf sizes were significantly smaller than the ones from the control group ([Km]=0mg/L) (Fig. 4 a,c, p < 0.001, df = 5, Krusal-Wallis rank sum test). When [Km]≥100mg/L, none of the germinated seedlings produced true leaves (Fig. 4 a, dark green line). Side roots are even more sensitive to increasing [Km]—the proportion of germinated seeds that produced side roots decreased to 0% as soon as [Km] reached 50mg/L (Fig. 4 a, violet red line; p < 0.001, logistic generalised linear model for binomial data, χ 2 = 594.17). The proportion of germinated seeds that fell down steadily increased with increasing [Km] (Fig. 4 a, brown line; logistic generalised linear model for binomial data, χ 2 = 84.621). As for the cotyledons, the proportion of bleached cotyledons drastically increased between [Km]=50mg/L and [Km]=100mg/L (Fig. 4 a, golden line; logistic generalised linear model for binomial data, χ 2 = 655.1). When [Km]≤50mg/L, less than 10% of germinated seedlings had bleached cotyledons; when [Km]≥100mg/L, more than 85% of germinated seedlings had bleached cotyledons. Besides, cotyledon size was also significantly smaller for [Km]≥100mg/L compared to the [Km] = 0 and 50 mg/L groups, while the [Km]=50mg/L group also had significantly smaller cotyledons compared to the [Km]=0mg/L group (Fig. 4 b, p < 0.001, df = 5, one-way nested ANOVA using linear mixed models). These measurements show that [Km]=100mg/L was the lowest concentration required for most negative effects on seeds to show a clear cut off compared to [Km]=50mg/L (Fig. 4 ). Therefore, I set [Km]=100mg/L as the threshold concentration to inhibit seed growth. 3.2-Direct somatic embryogenesis Different tissue types differ greatly in the ability to produce direct somatic embryos under their own 6-benzylaminopurine concentration ([BAP]) (Table 1 ; Fig. 5 ; p < 0.001, binomial generalised linear model, χ 2 = 58.30, df = 3). Shoot apical meristems (SAM) were significantly better at producing direct somatic embryos than other types of explants: nearly 80% of all SAM explants produced embryos after seven weeks in the light period (i.e. 4 weeks after finishing the kanamycin resistance test) and each explant produced from 2 ~ 50 embryos/explant (Table 1 ; Fig. 5 & 6 a). Some hypocotyls were also good at producing the embryos, but only ~ 10% of all hypocotyls could produce embryos ranging from 5 ~ 50 embryos/explant (Table 1 ; Fig. 5 & 6 b). Leaves were the worst at producing the embryos: only 1 leaf from three-week-old seedlings produced 1 tiny embryo (Fig. 6 c) and another leaf from six-week-old young plants produced two tiny embryos (Fig. 6 d). The embryos from SAMs were also best at rooting: more than 60% embryos cut from the original SAMs managed to produce roots ≥1cm in the rooting media, while only about 30% of embryos from the hypocotyls managed to do so and none of the leaf explants rooted (Table 1 ). When the embryos (regardless of where the embryos come from) successfully rooted in the rooting media (Fig. 6 f), more than 80% of them survived the whole acclimatising process and grew healthily in the glasshouse (Fig. 6 g-h). Table 1 Different tissue types’ ability for producing direct somatic embryos and their embryos’ rooting and surviving status during acclimitising process. Tissue type (include plate number) Number of explants that produced direct somatic embryos (n out of 14/plate)* Number of embryos per explant that produced direct somatic embryos (range in some cases)* Total number of embryos cut out from each type of explant† Total number of embryos from each tissue type that managed to root in rooting media† Total number of embryos from each tissue type that survived the whole acclimatising process† Hypocotyl 1 2 5–50/explant ~ 60 16 15 Hypocotyl 2 1 10 True leaves from 3-week-old seedlings 1 0 0 1 0 0 True leaves from 3-week-old seedlings 2 1 1 True leaves from 6-week-old young plants 1 1 2 2 0 0 True leaves from 6-week-old young plants 2 0 0 Shoot apical meristem 1 11 10–20 ~ 260 162 132 Shoot apical meristem 2 11 2–50 *These data were recorded 4 weeks after finishing kanamycin resistance test = 7 weeks into the light period. †These data were recorded 16 weeks after finishing kanamycin resistance test, when this direct somatic embryogenesis experiment finished. During this experiment, I noticed that the very base of some true leaves were left on the shoot apical meristems (SAM) (Fig. 1 d), as they were extremely hard to remove at the beginning of the kanamycin resistance test when the shoot of the seedling was only ~1cm tall. Therefore, these ‘leftovers’ of the true leaves grew at [BAP]=1mg/L. The ‘leftovers’ later grew bigger along with the main part of the SAMs and produced some embryos from the original cutting site (~ 15–20 embryos in total out of ~ 5 ‘leftovers’ I identified). Interestingly, both the ‘leftover’ leaves and their embryos (Fig. 6 e) seemed to survive much better than the leaves and the leaf-produced embryos that grew on [BAP] = 1.5mg/L medium (Fig. 6 c-d). ~10 of them even successfully developed into mature plants. I wondered whether the leaves may be healthier and survive better if I reduced the [BAP] to 1mg/L (i.e. same concentration as culturing hypocotyls and shoot apical meristems). Therefore, in a small follow-up experiment, I changed the BAP concentration to 1mg/L for all tissue types for direct somatic embryogenesis while other conditions remained the same. As was expected, the ability of direct somatic embryogenesis was greatly improved for true leaves from 3-week old seedlings—2 out of 16 explants (vs. original 2 out of 56) produced direct somatic embryos and the number of embryos per explant ranged from 5–50 (vs. original 1–2). Besides, if regenerated plantlets are transferred into a larger container with resupplied rooting medium, the plantlets can flower and set seeds in vitro in 3–6 weeks after being separated for rooting. These results show that hypocotyl, leaf and shoot apical meristem were all capable of producing direct somatic embryos despite huge differences. To maximise the efficiency and reduce total time of somatic embryogenesis, it would be better to use all materials from healthy 3-week-old seedlings (i.e. hypocotyl, cotyledon, true leaf and shoot apical meristem). 4. Discussion 4.1-Great potential for direct somatic embryogenesis in Portulaca amilis Portulaca amilis showed great potential in producing direct somatic embryos and it is relatively easy to induce direct somatic embryos and grow the embryos into mature plants (Table 1 ; Fig. 6 ). The whole regeneration process did not encounter the common bottlenecks that constrain somatic embryogenesis in other species, such as hard to induce direct somatic embryos, complex hormone combination, low survival of regenerated plantlets, long waiting time in various regeneration process (Litz and Gray, 1995 ; Park et al., 1998 ; Suzuki et al., 2004 ; Safdari and Kazemitabar 2010 ; Juturu et al., 2015 ). First, it is easy to propagate large numbers of sterile seedlings in vitro —the seeds are tiny (~ 0.5mm in diameter, Judd and Wunderlin, 1981 ) and can be thoroughly sterilised in large batches within 45 minutes using less hazardous reagents. Larger seeds (≥ 1mm, e.g. Portulaca oleracea ) usually require higher concentration of NaClO solution (~ 1.5–2.5% (m/m)) and some even require HgCl 2 , which are both more hazardous to both people and seeds (e.g. Bagheri et al., 2010 ; Raut et al., 2016 ; Sedaghati et al., 2019 , 2021 ). Second, it only takes 3 weeks for the sterile seedlings to be ready as explant sources for direct somatic embryogenesis. These explants need no extra sterilisation compared to explants from non-sterile plants. For example, stem and leaf cuttings from non-sterile plants often need to be washed, sterilised with either 70 ~ 75% ethanol, ~ 1% NaClO or 0.1% HgCl 2 and properly dried in the flow hood (e.g. Portulaca grandiflora and P. pilosa , Safdari and Kazemitabar 2010 ; Chen et al., 2020 ). Third, the hormone combinations for inducing large numbers of direct somatic embryos in P. amilis is relatively simple compared to its relatives such as P. oleracea , P. pilosa and P. grandiflora . (Safdari and Kazemitabar 2010 ; Sedaghati et al., 2019 ; Chen et al., 2020 ). In P. amilis , 1mg/L of 6-benzylaminopurine (BAP) is sufficient to induce large numbers of direct somatic embryos from hypocotyl, leaves and shoot apical meristems (Table 1 ; Fig. 6 )—all parts of the shoot in a three-week-old seedling can produce direct somatic embryos, which is not common in other species. For P. oleracea , stems cannot produce direct somatic embryos and must go through the callus phase, while leaves need both BAP and 1-naphthaleneacetic acid (NAA) under a certain combination to produce large numbers of direct somatic embryos (Sedaghati et al., 2019 ). For P. pilosa , the leaves need both thidiazuron (TDZ) and NAA to produce direct somatic embryos (Chen et al., 2020 ). For P. grandiflora , the leaves cannot produce direct somatic embryos and require both BAP and NAA to induce somatic embryos from callus (Safdari and Kazemitabar 2010 ). Lastly, when the embryos of P. amilis successfully rooted in the rooting medium (Fig. 5 f), one can acclimatise them easily in large batches in a growth cabinet with > 80% survival and hardly any abnormal development, as long as the soil is moist and well-drained (Fig. 2 ). These four advantages in direct somatic embryogenesis, plus the advantages of being a diploid and having a fully sequenced genome, make Portulaca amilis a good model species for future studies, including transformation (Danin et al., 1978 ; Judd and Wunderlin, 1981 ; Walter et al., 2015 ; Gilman et al., 2022 ). To facilitate future studies involving direct somatic embryogenesis of P. amilis , I hereby provide an optimised protocol based on result section 3.2 (Table 2 ), which is simple, efficient and economical for batch propagation or maintain large quantity of sterile plant lines. The main procedure include (1) sterilising seeds; (2) plate and grow seeds into 3-week-old seedlings; (3) cut the seedlings into hypocotyl, cotyledon, true leaves and shoot apical meristem; (4) plate and culture in dark period (range from 1-maximum 3 weeks); (5) culture in light period to induce direct somatic embryos and grow them into ≥5mm mini plantlets (range from 4–8 weeks); (6) separate the mini plantlets for rooting (1–2 weeks); (7.1) keep rooted plantlets growing in sterile environment to flower and set seeds (2–4 weeks), or (7.2) acclimatise rooted plantlets in sterile water (3–4 days) and then sterile soil (7–12 days) in growth chamber; (8) acclimatise rooted plantlets in non-sterile soil in the glasshouse (3–4 weeks to obtain full-sized plants setting seeds). The whole process from sterilising seeds to regenerated mature plants setting seeds (sterile/non-sterile) takes only 3–4 months, while from explants to moving plantlets to soil already takes nearly 2 months in P. oleracea (Sedaghati et al., 2019 ). Besides, the acclimatisation setting in step 6–7 can be reused and takes little space due to smaller plant size, thus highly economical. Table 2 Optimised protocol for direct somatic embryogenesis. procedure medium/soil condition growth/treatment condition total timing 1. sterile seeds (1)70% ethanol: 1 min; (2)1 Milton mini tablet sterilised water: 25 min; (3)sterile water: 3 times, 1min/time sterile environment 30-40min 2. plate and grow seeds into 3-week-old seedlings full strength MS with Gamborg B5 vitamin; 3% (m/v) sucrose; 0.7% (m/v) bacto-agar; pH = 5.8 ± 0.05* temperature: 25°C; light intensity: 5300 Lux; photoperiod: 16h light/8h dark; sterile environment 3 weeks 3. cut the seedlings into hypocotyl, cotyledons, true leaves and shoot apical meristem cut on thin layer of sterile water to avoid dehydration sterile environment 1–2 hours depending on quantity 4. plate and culture explants in darkness full strength MS with Gamborg B5 vitamin; 3% (m/v) sucrose; 0.7% (m/v) bacto-agar; 1mg/L BAP; pH = 5.8 ± 0.05* temperature: 25°C; light intensity: 0 Lux (complete darkness) 1-maximum 3 weeks† 5. plate and culture explants in light to induce direct somatic embryos and grow them into ≥5mm plantlets full strength MS with Gamborg B5 vitamin; 3% (m/v) sucrose; 0.7% (m/v) bacto-agar; 1mg/L BAP; pH = 5.8 ± 0.05*; refresh media once every 2–3 weeks to keep inducing embryos temperature: 25°C; light intensity: 5300 Lux; photoperiod: 16h light/8h dark; sterile environment 4–8 weeks 6. cut and root regenerated plantlets until roots reach 1-2cm full strength MS with Gamborg B5 vitamin; 3% (m/v) sucrose; 0.7% (m/v) bacto-agar; pH = 5.8 ± 0.05* temperature: 25°C; light intensity: 5300 Lux; photoperiod: 16h light/8h dark; sterile environment 1–2 weeks 7.1 keep growing in sterile environment until fruiting full strength MS with Gamborg B5 vitamin; 3% (m/v) sucrose; 0.7% (m/v) bacto-agar; pH = 5.8 ± 0.05*; refresh media once every 2–3 weeks to keep plantlets growing temperature: 25°C; light intensity: 5300 Lux; photoperiod: 16h light/8h dark; sterile environment 2–4 weeks 7.2 acclimatise rooted plantlets in sterile water, then sterile soil in growth chamber (1) sterile water in 2ml centrifuge tubes: 3–4 days, refill with water if water level is low; (2) sterile soil in small pots (M3 soil:sand:perlite = 2:2:1): 7–12 days, water once a day or once every two days based on soil moisture temperature: 25°C; light intensity: 5300 Lux; photoperiod: 16h light/8h dark; sterile to non-sterile environment; centrifuge tubes and small pots kept in 1000µl pipette tip boxes with lids gradually opening 10–16 days 8. acclimatise plantlets in glasshouse (after 7.2) non-sterile soil in medium-large pots (0.2-0.5L); M3 soil: sand: perlite = 2:2:1; once a day or once every two days based on soil moisture temperature: 18–25°C; light intensity: ~5300 Lux, vary a bit based on temperature; photoperiod: 16h light/8h dark; non-sterile environment 3–4 weeks for full sized plants setting seeds *adjusted by 0.5mol/L KOH and 0.5mol/L HCl †1 week is sufficient to induce direct somatic embryos based on a small follow-up experiment. Depending on the purpose, dark period can be longer, but no longer than 3 weeks due to reduced explant survival rate. 4.2-Threshold kanamycin (Km) concentration for Portulaca amilis The threshold kanamycin concentration ([Km]) for Portulaca amilis is 50mg/L for tissues and 100mg/L for seeds, much lower than the threshold [Km] for tissues and seeds of Portulaca oleracea (both 250mg/L, Sedaghati et al., 2019 , 2021 ). Despite from the same genus, two species respond to Km differ greatly. For the four tissue types of P. amilis I used, all regeneration events, from callus to direct somatic embryos, completely disappeared when [Km] was only 50mg/L (Fig. 3 b-c). But for P. oleracea , regeneration only stopped when [Km] reached 250mg/L for leaves—five times the threshold [Km] of P. amilis (Sedaghati et al., 2019 ). For the seeds, germination was not inhibited by increasing [Km] in P. amilis —more than 90% of all seeds still germinated normally on [Km]=250mg/L (Fig. 4 a, light green line). I therefore used several other parameters (e.g. the proportion of germinated seeds that produce true leaves) to comprehensively determine threshold [Km] (see Result 3.1.1). In P. oleracea , however, germination rate decreased with increasing [Km] and no seeds germinated at 250mg/L, making the threshold [Km] obvious (Sedaghati et al., 2021 ). The tissues of Portulaca amilis are extremely sensitive to Km while the seeds are less so compared to other species. Past studies show that the threshold [Km] for leaf or stem explants ranges from 15mg/L to 250mg/L, above which either regeneration stops completely or the explants all died (Filatti et al., 1987; Bagheri et al, 2010 ; Bahmanker et al., 2015, Sedaghati et al., 2019 ). While the tissues of P. amilis stay near the lower boundary of threshold [Km], the tissues of many species fall between 100mg/L-200mg/L, such as honeydews ( Cucumis melo ), pigeon peas ( Cajanus cajan ) and Physalis pruinosa (e.g. Ren et al., 2012 ; Raut et al., 2016 ; Swartwood and Van Eck, 2019 ). For seeds, the threshold [Km] ranges from 75mg/L to 250mg/L and seeds of P. amilis lie in the middle (Gatzek et al., 2002 ; Darqui et al., 2018 ; Niazian et al., 2019 ; Sedaghati et al., 2021 ). These threshold [Km] were chosen either based on germination rate, if germination rate decreases with increasing [Km], or other criteria such as proportion of germinated seeds that produced side roots, if germination rate was not negatively affected by increasing [Km]. As we can see, the threshold [Km] depends on the species and tissue/organ types and is highly variable even for closely related species (e.g. Portulaca amilis vs. P. oleracea ). Therefore, if one decides to use kanamycin as the selection reagent for transforming a new species, variant or tissue/organ type, they must perform a kanamycin resistance test ahead of time. Otherwise, a too low [Km] may cause ‘false positive’ results (i.e. non-transformed plants survive/regenerate on selective media) while a too high [Km] may kill everything, no matter whether the tissues/organs are successfully transformed or not (e.g. Sedaghati et al., 2019 , 2021 ). 4.3-Outlook and conclusion Despite various advantages in direct somatic embryogenesis of Portulaca amilis under the above experimental procedure, it is worthwhile investigate other aspects in the regeneration process that may be critical to future experimental setups. First, tissues from young seedlings are potentially good explant source, as actively growing tissues have more endogenous hormones and require less external ones for somatic embryogenesis (Deo et al., 2010 ; Kim et al., 2010 ; Sedaghati et al., 2019 ). Apart from Portulaca amilis , several other species use young tissues like hypocotyl, cotyledons, germinating seeds and immature zygotic embryos to produce somatic embryos (e.g. Gossypium , Solanum lycopersicum , Zea mays , Arabidopsis ; Fillatti et al., 1987 ; Su et al., 2009 ; Jurutu et al., 2015; Lowe et al., 2018 ; Ikeuchi et al., 2019 ). Second, a few stress factors can positively or negatively affect somatic embryogenesis. The two critical ones are wounding and antibiotic toxicity, which require particular attention as somatic embryogenesis is often involved in Agrobacterium- mediated transformation to knockout or overexpress genes of interest (Anami et al., 2013 ; Ikeuchi et al., 2019 ). Wounding induces hormone synthesis and in general promotes somatic embrygenesis (Ikeuchi et al., 2016 , 2019 ; Mozgová et al., 2017 ). On the other hand, various antibiotics often inhibit somatic embryogenesis even for the ones that do not kill cells like cefotaxime (i.e. the most commonly used antibiotics to remove Agrobacterium after transformation; Ling et al., 1998 ; Teixeira da Salva and Fukai, 2001). In Portualca amilis , my small pilot experiment (Zhao, 2025 ) showed that direct somatic embryogenesis was greatly inhibited when the tissues are exposed to moderate concentration of cefotaxime for only two hours (150mg/L, commonly used in transformation; Ling et al., 1998 ; Teixeira da Salva and Fukai, 2001; Sedaghati et al., 2019 , 2021 ). Therefore, although P. amilis showed great direct somatic embryogenesis ability under benign conditions, one need to investigate whether such ability can be maintained in successfully transformed cells. In conclusion, this study demonstrated a simple, efficient way to batch propagate direct somatic embryos of Portulaca amilis and also reported kanamycin resistance ability for tissue and seeds. The results can be applied to various future studies involving direct somatic embryogenesis, especially towards developing a successful transformation protocol in the species. Abbreviations BAP 6-benzylaminopurine HCl hydrochloric acid Km kanamycin KOH potassium hydroxide MS medium Murashige and Skoog medium (MS) Declarations Acknowledgements: I would like to thank Cambridge International Trust and Newnham College for funding my PhD (tuition and stipend), part of which gave rise to this manuscript. I would like to thank the following persons and teams for providing me with technical and data analysis support, access to facilities and manuscript revision suggestions (in alphabetical order of last names and organisations): Dr. Julie Bailey, Dr. Sam Brockington, Professor Kate Fleet, Professor Beverley Glover, Fiona Holder, Professor Nik Cunniffe (special thanks for statistical analysis advice), Dr. Jiafu Tan (special thanks for tissue culture advice), Dr. Xiaoyu Wang; Cambridge University Botanic Garden horticultural team, Newnham College, Sainsbury Lab security, reception, administrative and technical staff. Statements and Declarations: This work was supported by Cambridge International & Newnham College Scholarship funded by the Cambridge Commonwealth, European & International Trust. The author has no relevant financial or non-financial interests to disclose. Conflict of interest: The author has no relevant financial or non-financial interests to disclose. Author contribution statement: I am the only author for the manuscript, responsible for experimental design, data collection, analyses and interpretation, manuscript writing and publishing. Ethics declaration: Not applicable. Supplementary information: Apart from data directly presented in the tables, all spreadsheets and R code (version 4.2.1, detailed comments included) used for data analyses in this manuscript are included in supplementary information as an .xlsx file (compiled raw data) and .rmd file (compiled R code) for running the code with the data in R. Data availability statement: All raw data and code included in supplementary information. References Anami, S., Njuguna, E., Coussens, G., Aesaert, S. and Van Lijsebettens, M. (2013). Higher plant transformation: principles and molecular tools. The International Journal of Developmental Biology 57: 483-494. Bagheri, K., Javaran, M.J., Mahboudi, F., Moeini, A. and Zebarjadi, A. (2010). 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South African Journal of Botany 140: 1-3. Zhao, Y. (2025). Towards a systematic analysis and comparison of perianth evolution in Caryophyllineae in the context of the synflorescence [Apollo - University of Cambridge Repository]. https://doi.org/10.17863/CAM.122697 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8790648","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":589916006,"identity":"7257e42a-e2aa-4528-8b4a-e29e3db82611","order_by":0,"name":"Yi Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIie3RMQrCMBSA4RcCTq2uikM8QovgJF29RqBQFwdHwYKVgqOugpeoCM6VB3V0c8miNygIjmKaIjjY1lEwPyHDIx8JBECn+8WoWuA05BZf1ITO42+I2wok4WpCgnKSKyBRdkwRqCDWEeht7Pdp94xXecuesUFGfKeYIEB3nXj1nvAsSYS9xYwkbilxjRrSnuCKcDuUhAS0lKDxQLLbDNN3MisjJDQXSKL2KL+FUUWwkLSQhNRcem5TjMYxt4QdZYQnx0JSPyHejHvfaWyGuzSdCMZWeLik/rSQdOQb3t8pVwyvD/ocC6onOp1O9+89AXcSWaBj0IivAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4433-1712","institution":"University of Cambridge","correspondingAuthor":true,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2026-02-04 22:57:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8790648/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8790648/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102821733,"identity":"34dbd825-bfd3-442f-ba6b-3c9b29ecdf07","added_by":"auto","created_at":"2026-02-17 07:44:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":586400,"visible":true,"origin":"","legend":"\u003cp\u003ePhotos and schematic diagrams of \u003cem\u003ePortulaca amilis\u003c/em\u003e (Portulaceae) (a) a mature flower. (b) a mature plant setting seeds. (c) 3-week-old sterile seedlings on a petri dish. (d) Schematic diagram of a 3-week-old seedling (drawn in Microsoft Paint). This diagram indicates the average size of a 3-week-old seedling. The shoot of the seedlings usually varies from 0.6-1.2cm. Red dashes—cutting points to separate each tissue. Due to size restriction and concerns on survival, the ‘shoot apical meristem’ used in this chapter was a short stem (~3mm) that starts from the real shoot apical meristem to the cutting point between the pair of cotyledons, and the base of the true leaves were sometimes left on the stem.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8790648/v1/ff5e2e064342fa9eb8bee071.png"},{"id":102963113,"identity":"ebe19323-7830-491a-aad4-084c352d456e","added_by":"auto","created_at":"2026-02-19 04:13:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":190183,"visible":true,"origin":"","legend":"\u003cp\u003eSetups for acclimatising rooted regenerated plantlets. (a) rooted plantlets in sterile water. (b) rooted plantlets in soil. Schematic diagrams drawn in Medibang Paint Pro version 2.7.21.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8790648/v1/a797b86c0784ea2f72bfecfc.png"},{"id":102963241,"identity":"9b4dd481-ede5-4921-8ca2-01fe3130d8c0","added_by":"auto","created_at":"2026-02-19 04:14:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":168580,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent tissue types’ responses to increasing kanamycin concentration. (a) survival. (b) ability to produce callus. (c) ability to produce direct somatic embryos. (d) bleaching. Different letters on each graph indicate that there were significant differences among tissue types based on post-hoc tests (TukeyHSD).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8790648/v1/6cb613b28e476b9adb4faad3.png"},{"id":102963252,"identity":"ea279b1f-7612-446a-8972-6ff8d4827aeb","added_by":"auto","created_at":"2026-02-19 04:14:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":88785,"visible":true,"origin":"","legend":"\u003cp\u003eHow increased kanamycin concentration ([Km]) affected different aspects of seed growth. (a) how [Km] affected proportion of seeds that germinated etc. (see legends); (b) how [Km] affected relative cotyledon size (mm\u003csup\u003e2\u003c/sup\u003e); (c) how [Km] affected relative first true leaf size (mm\u003csup\u003e2\u003c/sup\u003e). Different letters indicate that there were significant differences among different [Km] groups based on post-hoc tests (TukeyHSD for b, Wilcoxon rank sum test for c). There were 3 plates for each [Km] and 50 seeds on each plate. For cotyledon and true leaf size, N=10 for each plate and 10×3=30 for each [Km]. Each dot of the same colour represents a plate in (a). There were some dots that completely overlapped in (a).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8790648/v1/facb2cb8d0af3ef01ddff7d6.png"},{"id":102963104,"identity":"37f80584-ebd6-47ff-b5f4-25f67edab7bb","added_by":"auto","created_at":"2026-02-19 04:13:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":54794,"visible":true,"origin":"","legend":"\u003cp\u003eThe proportion of total tissue that produced direct somatic embryos for each tissue type. Different letters indicate that there were significant differences among tissue types based on post-hoc tests (TukeyHSD). Note that the two data points for shoot apical meristem completely overlapped in this case. Data was recorded 4 weeks after kanamycin resistance test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8790648/v1/78b8e84244f15d3d291c4536.png"},{"id":102963261,"identity":"b00754aa-0802-44da-9c84-68fb0ebb083f","added_by":"auto","created_at":"2026-02-19 04:14:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1213131,"visible":true,"origin":"","legend":"\u003cp\u003eDirect somatic embryogenesis using tissues from control group of kanamycin resistance test. (a) embryos from a hypocotyl; (b) embryos from a shoot apical meristem; (c) an embryo (red circle) from a true leaf of a 3-week-old seedling; (d) 2 embryos from a true leaf of a 6-week-old young plant; (e) embryos from a ‘leftover’ leaf (white circle) on a shoot apical meristem; (f) a successfully rooted embryo from a hypocotyl. Due to a typo, the date was 2022.11.30 instead of 2023.11.30; (g) seedlings from direct somatic embryos just before moving to the glasshouse; (h) grown up plants from direct somatic embryos in the glasshouse. Red arrows—the hard, green tissues where the direct somatic embryos emerge.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8790648/v1/31521e6efad7af0900c2d14c.png"},{"id":106705177,"identity":"06cfdd72-5f4d-475c-ae20-cdd21746ae99","added_by":"auto","created_at":"2026-04-12 08:40:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3797040,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8790648/v1/67ffaef2-3252-4339-9f63-d99e181d8269.pdf"}],"financialInterests":"","formattedTitle":"A simple way to batch propagate direct somatic embryos and kanamycin resistance of Portulaca amilis (Portulacaceae)","fulltext":[{"header":"Key message","content":"\u003cp\u003eDirect somatic embryogenesis occurs in young shoot tissues of with 1mg/L BAP on full MS medium. Kanamycin resistance threshold is 50mg/L for shoot tissues and 100mg/L for seeds.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003ePlant somatic embryogenesis has many critical applications in science, agriculture and industry, such as plant breeding, improving crop yield, medicine production, gene transformation and testing gene functions (Raemakers et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Birch, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Hansen and Wright, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Newell, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Kamle et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Sedaghati et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the past few decades, people have reported at least 200 species in seed plants that are capable of direct to indirect somatic embryogenesis, varying degrees on a spectrum (Williams and Maheswaran, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Wann, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Carman, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Raemakers et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). The key distinction between direct and indirect somatic embryogenesis (i.e. two extremes on the direct-indirect spectrum) is whether the tissue undergoes the callus phase before producing embryos (Sharp et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Evans et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). And due to the callus phase and tissue re-differentiation, it generally takes much longer for indirect somatic embryogenesis within a species (vary from weeks to years, e.g. Davidonis and Hamilton, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Sedaghati et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSomatic embryogenesis can sometimes be difficult to induce and optimise, as there are numerous factors involved. The main factors involved are tissue types, species (include specific genotype/varieties) and growth hormones (e.g. Fillatti et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Lowe et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sun et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sedaghati et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For example, the optimised protocol for cotton (\u003cem\u003eGossypium\u003c/em\u003e) and tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) are both via indirect somatic embryogenesis using hypocotyl or cotyledon (Fillatti et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Godishala et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Juturu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In \u003cem\u003ePortulaca oleracea\u003c/em\u003e, leaves can produce both direct and indirect somatic embryos while stems can only produce indirect ones under similar hormone combinations (Sedaghati et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). 2,4-dichlorophenoxyacetic acid (2,4-D), a synthetic auxin, has higher chance to induce abnormal somatic embryos in various crops or ornamental plants than other auxin or cytokinin derivatives (reviewed in Garcia et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Apart from these factors, explant loss occurs in almost each step of somatic embryogenesis, from initial explant preparation (including sterilisation), embryogenesis to final acclimatisation and growth of somatic embryos to mature plants, so the final survival rate of mature plants can be low (Litz and Gray, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Park et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Therefore, optimising conditions for somatic embryogenesis can be time consuming and highly species dependent, yet a successful protocol will benefit numerous downstream research.\u003c/p\u003e \u003cp\u003eIn this study, I established an efficient, simple way to induce, regenerate and acclimatise direct somatic embryos from various young shoot tissues in \u003cem\u003ePortulaca amilis\u003c/em\u003e (Portulacaceae) with high survival rate, along with its kanamycin resistance ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The regenerated plantlets can be maintained in sterile environment and set seeds \u003cem\u003ein vitro\u003c/em\u003e or acclimatised and set seeds in non-sterile environment, flexible for different purposes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMembers of \u003cem\u003ePortulaca\u003c/em\u003e produce betalain, use C4-CAM photosynthesis, can be consumed as vegetables or medical herbs, and has unusual perianth evolutionary history (Kubitzki et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Guralnick and Jackson, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Brockington et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Sedaghati et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). They have great potential for biochemical, physiological, medical, genetical and morphological studies, and it is thus worth establishing a model species within this genus. \u003cem\u003eP. amilis\u003c/em\u003e is potentially a better candidate model species than closely-related, well-studied species like \u003cem\u003eP. oleracea\u003c/em\u003e, \u003cem\u003eP. grandiflora\u003c/em\u003e and \u003cem\u003eP. pilosa\u003c/em\u003e, despite all three having direct or indirect somatic embryogenesis/organogenesis protocols (Safdari and Kazemitabar, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Sedaghati et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Xiong et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). First, apart from fast growth cycle and massive seed production shared by all \u003cem\u003ePortulaca\u003c/em\u003e, \u003cem\u003eP. amilis\u003c/em\u003e is a simple diploid instead of species complexes like \u003cem\u003eP. oleracea\u003c/em\u003e and \u003cem\u003eP. grandiflora\u003c/em\u003e, more suitable for gene knock-out (Danin et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Mishiba and Mii, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Walter et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Second, the seeds are highly viable in long-term lab storage instead of low survival rate like \u003cem\u003eP. pilosa\u003c/em\u003e (Kim and Carr, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), easier to maintain large lab populations. Third, \u003cem\u003eP. amilis\u003c/em\u003e is the only \u003cem\u003ePortulaca\u003c/em\u003e with a fully sequenced genome, available to refer and annotate gene functions (Gilman et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Such advantages make \u003cem\u003eP. amilis\u003c/em\u003e a better option for transgenic analyses on gene functions, especially genes related to betalain pathway and unusual perianth evolution, both of which are active research areas (Brockington et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Sheehan et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, transgenic analyses usually involve designing gene editing constructs (e.g. CRISPR) to silence, knockout or overexpress certain genes and searching for off-target effects, where somatic embryogenesis is required in selecting and regenerating successfully transformed individuals. As a result, the findings of this study on direct somatic embryogenesis and kanamycin resistance of \u003cem\u003eP. amilis\u003c/em\u003e will benefit future experimental design in transformation, genetic analyses, mass propagation and medical applications of the species.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003eSince direct somatic embryogenesis is more efficient than the indirect one and can be highly tissue-dependent, I tested different shoot tissues\u0026rsquo; ability on direct somatic embryogenesis in \u003cem\u003ePortulaca amilis\u003c/em\u003e. Besides, as direct somatic embryogenesis is closely associated with plant transformation, which often use kanamycin as the selection agent in \u003cem\u003eAgrobacterium-\u003c/em\u003emediated transformation (e.g. Fillatti et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Gatzak et al., 2002; Bagheri et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Raut et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sedaghati et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), I tested both seed and different tissues\u0026rsquo; ability on kanamycin resistance to prepare for future transformation experiments via tissue culture or seeds. The main experimental procedures were inspired by protocols of Sedaghati et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) on tissue and seed transformation of \u003cem\u003eP. oleracea\u003c/em\u003e, as \u003cem\u003eP. oleracea\u003c/em\u003e is closely related to \u003cem\u003eP. amilis\u003c/em\u003e. All \u003cem\u003eP. amilis\u003c/em\u003e material used in this study originally came from United States, Department of Agriculture (PI 677126).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1-Explant preparation\u003c/h2\u003e \u003cp\u003eI sterilised\u0026thinsp;~\u0026thinsp;1200 seeds with 70% ethanol for one minute, then with Milton Mini Tablet-sterilised water for 25 minutes (solution contains\u0026thinsp;~\u0026thinsp;0.02% (m/m) sodium dichloroisocyanurate, 1 tablet in ~\u0026thinsp;50ml sterile water), and finally washed the seeds three times with sterile water for 1 minute/time. ~200 seeds were prepared for tissue kanamycin test and direct somatic embryogenesis while the rest\u0026thinsp;~\u0026thinsp;1000 were prepared for seed kanamycin test.\u003c/p\u003e \u003cp\u003eFor tissue kanamycin test and direct somatic embryogenesis, the sterile seeds were plated on full strength Murashige and Skoog (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) medium (MS) with Gamborg B5 vitamins, supplemented with 3% (m/v) sucrose and 0.7% (m/v) bacto-agar. The pH of the medium was adjusted to 5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 by 0.5 mol/L KOH and 0.5 mol/L HCl before autoclaving. I germinated and grew these seeds in a growth chamber (Panasonic MLR-352-PE Climate Chamber), and set the conditions to 25\u0026deg;C, with 16h light/8h dark photoperiod and light intensity of ~\u0026thinsp;5300 Lux. When the seedlings reached three weeks old, I cut and separated each seedling into three explant types: hypocotyl, true leaves and shoot apical meristem (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-d). I kept\u0026thinsp;~\u0026thinsp;30 seedlings growing for another three weeks and collected young leaves (~\u0026thinsp;5-7mm in length) from these six-week-old plants as the fourth explant type. I used all four explant types in the kanamycin resistance test and direct somatic embryogenesis.\u003c/p\u003e \u003cp\u003eFor seed kanamycin test, the sterile seeds were soaked in a 10ml sterile conical flask filled with sterile liquid medium (full strength MS with Gamborg\u0026rsquo;s B5 vitamins, supplemented with 3% (m/v) sucrose, pH\u0026thinsp;=\u0026thinsp;5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05). I sealed the flask and shook it gently (120rpm) in darkness at room temperature for 24\u0026thinsp;~\u0026thinsp;36h before the seeds were ready for plating. The soaking mimics the environment before and during seed transformation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2-Setups for kanamycin (Km) resistance test\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1-Tissue kanamycin test\u003c/h2\u003e \u003cp\u003eTo find out the threshold kanamycin concentration ([Km]) that inhibits various tissue growth, I set up a kanamycin concentration gradient ([Km]\u0026thinsp;=\u0026thinsp;0 (control), 50, 100, 150, 200, 250mg/L) on full strength MS medium supplemented with 3% (m/v) sucrose, 0.7% (m/v) bacto-agar, 1mg/L (hypocotyl and shoot apical meristem) or 1.5mg/L (leaf) of 6-benzylaminopurine (BAP), pH\u0026thinsp;=\u0026thinsp;5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05. For each explant type under a certain [Km], I had two plates as biological replicates and each plate had 14 explants. Therefore, there were 14 explants/plate \u0026times; 2 plates/(tissue type \u0026times; concentration group) \u0026times; 4 tissue types \u0026times; 6 concentration groups\u0026thinsp;=\u0026thinsp;672 explants. The explants were cultured on the corresponding medium in the growth chamber at 25\u0026deg;C, first in complete darkness for three weeks (dark period), then in 16h light/8h dark photoperiod with light intensity of ~\u0026thinsp;5300 Lux for another three weeks (light period). I resupplied the explants with fresh media once every three weeks.\u003c/p\u003e \u003cp\u003eBy the end of the light period (the sixth week from putting freshly-cut explants on plates), I recorded the number of survived explants on each plate, described their colour (i.e. bleached/unbleached), and the number of explants that produced callus and direct somatic embryos. I used generalised linear modelling and analysis of covariance for binominal data in R (version 4.2.1) to look for the threshold kanamycin concentration that inhibits explant growth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2-Seed kanamycin test\u003c/h2\u003e \u003cp\u003eTo find out the threshold kanamycin concentration ([Km]) that inhibits seed growth, I set up a kanamycin concentration gradient ([Km]\u0026thinsp;=\u0026thinsp;0 (control), 50, 100, 150, 200, 250mg/L) on full strength MS medium with Gamborg\u0026rsquo;s B5 vitamins, supplemented with 3% (m/v) sucrose, 0.7% (m/v) bacto-agar, pH\u0026thinsp;=\u0026thinsp;5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05. For each Km concentration, I had three plates and I plated 50 seeds/plate. Therefore, I had 3 plates/concentration group \u0026times; 6 concentration groups\u0026thinsp;=\u0026thinsp;18 plates, and 18 plates \u0026times; 50 seed/plate\u0026thinsp;=\u0026thinsp;900 seeds in total. I grew the seeds in the growth chamber under 16h light/8h dark photoperiod (light intensity of ~\u0026thinsp;5300 Lux) at 25\u0026deg;C for three weeks.\u003c/p\u003e \u003cp\u003eBy the end of the third week, I recorded several parameters for each plate, including the number of seeds that germinated, produced true leaves, produced side roots, fell down and were bleached after kanamycin treatment. I also randomly chose 10 germinated seeds from each plate and estimated the relative size of their cotyledons and first true leaves by measuring the length and width of each leaf and calculating the product of these two parameters. I analysed these data using either generalised linear modelling for binomial data, or one-way (nested) ANOVA under either parametric or non-parametric assumptions in R (version 4.2.1).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3-Direct somatic embryogenesis and acclimatisation for somatic embryos\u003c/h2\u003e \u003cp\u003eSince the control group of tissue kanamycin test ([Km]=0mg/L) shared the same growth condition as direct somatic embryogenesis, after the kanamycin resistance test, I used all surviving explants in the control group to test the ability of various tissues to produce direct somatic embryos. To induce direct somatic embryos, I continued to keep these explants under the same temperature and light conditions as the \u0026lsquo;light period\u0026rsquo; in the kanamycin resistance test, and resupplied them with fresh control group media once every three weeks. Once the diameter of the embryos reached \u0026ge;5mm and became mini plantlets, I cut the regenerated plantlets out of the original explants and put them in rooting medium without BAP (full strength MS medium supplemented with 3% (m/v) sucrose, 0.7% (m/v) bacto-agar, pH\u0026thinsp;=\u0026thinsp;5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05) under the same temperature and light conditions.\u003c/p\u003e \u003cp\u003eWhen the roots of the regenerated plantlets reached about 1\u0026thinsp;~\u0026thinsp;2cm, I gradually acclimatised the plantlets to a non-sterile environment with fluctuating temperature. I took the rooted plantlets out of the media, washed away the media with sterile water and grew them in 2ml centrifuge tubes filled with sterile water for three to four days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). All centrifuge tubes were kept in a clean 1000\u0026micro;l pipette tip box with the box lid closed in the growth chamber (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Then I moved these rooted plantlets from sterile water to sterile soil in 3\u0026times;3\u0026times;4cm small pots (M3 soil:sand:perlite\u0026thinsp;=\u0026thinsp;2:2:1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). I also kept the pots in clean 1000\u0026micro;l pipette tip boxes in the growth chamber and gradually opened the box lid to acclimatise the rooted plantlets to a non-sterile environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). These rooted plantlets were watered once a day or once every two days based on soil moisture. When the rooted plantlets had acclimatised in the soil for more than a week and reached about 3-5cm in height, I moved them to the glasshouse (16h light/8h dark photoperiod, light intensity\u0026thinsp;~\u0026thinsp;5300 Lux but varied a bit based on weather, temperature 18\u0026thinsp;~\u0026thinsp;25\u0026deg;C) and transplanted them to larger pots in the same soil (non-sterile). These plants were watered by Cambridge University Botanic Garden staffs on a regular basis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn the 10th week from putting freshly cut explants on plates (i.e. four weeks after kanamycin resistance test/seven weeks into light period), I recorded the type and the number of explants that directly produced somatic embryos, and the approximate number of embryos produced by a single explant. By the end of the whole experiment (i.e. 16 weeks after the kanamycin resistance test), I recorded the total number of mini plantlets that were cut out from each type of explant, the source (i.e. which explant type the plantlet originated from) and number of plantlets that successfully rooted in the rooting medium, and the source and number of regenerated plantlets that survived the whole acclimatising process. For this set of data, I either used generalised linear models for binominal data in R (version 4.2.1) or directly summarised the data into tables to look for which types of tissue are better at surviving and producing direct somatic embryos under control conditions ([Km]=0mg/L).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1-Kanamycin (Km) resistance test\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1-Tissue Km threshold\u003c/h2\u003e \u003cp\u003eAs soon as kanamycin (Km) was present at 50mg/L, all tissue types were more or less negatively affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The proportion of surviving true leaves from both 3-week-old seedlings and 6-week-old plants decreased quickly with increased Km concentration ([Km]), while the survival of hypocotyl and shoot apical meristem (SAM) were less negatively affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea; analysis of covariance (ANCOVA) for binomial data; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for both [Km]\u0026mdash;χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;16.88 and tissue type\u0026mdash; χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;557.98, df\u0026thinsp;=\u0026thinsp;3). At [Km]=0mg/L, both types of true leaves were significantly better at producing callus, while hypocotyl and SAM were significantly better at producing direct somatic embryos, and these regeneration activities soon predicted to decrease to 0 when [Km]\u0026ge;50mg/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb,c; ANCOVA for binomial data; for callus, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for both [Km]\u0026mdash;χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;123.29 and tissue type\u0026mdash;χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;83.45, df\u0026thinsp;=\u0026thinsp;3; for direct somatic embryos, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for [Km]\u0026mdash;χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;29.16, p\u0026thinsp;=\u0026thinsp;0.0064 for tissue type\u0026mdash;χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;12.30, df\u0026thinsp;=\u0026thinsp;3). Moreover, the proportion of bleached tissues quickly increased from 0% to 100% for all tissue types when [Km]\u0026ge;50mg/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed; ANCOVA for binomial data; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for [Km] and non-significant for tissue type).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on these results, the threshold kanamycin concentration is 50mg/L for various tissue type from seedlings or young plants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1-Seed Km threshold\u003c/h2\u003e \u003cp\u003eOverall, kanamycin (Km) had strong negative effects on all recorded parameters except seed germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Seed germination rate slowly increased with increasing Km concentration ([Km]) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, light green line; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, logistic generalised linear model for binomial data, χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;26.71). On the other hand, the proportion of germinated seeds that produced true leaves quickly decreased with increasing [Km] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, dark green line; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, logistic generalised linear model for binomial data, χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;538.39)\u0026mdash;almost all germinated seeds produced true leaves when there was no Km, while only\u0026thinsp;~\u0026thinsp;40% of germinated seeds produced true leaves under [Km]=50mg/L and their true leaf sizes were significantly smaller than the ones from the control group ([Km]=0mg/L) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,c, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, df\u0026thinsp;=\u0026thinsp;5, Krusal-Wallis rank sum test). When [Km]\u0026ge;100mg/L, none of the germinated seedlings produced true leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, dark green line). Side roots are even more sensitive to increasing [Km]\u0026mdash;the proportion of germinated seeds that produced side roots decreased to 0% as soon as [Km] reached 50mg/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, violet red line; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, logistic generalised linear model for binomial data, χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;594.17). The proportion of germinated seeds that fell down steadily increased with increasing [Km] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, brown line; logistic generalised linear model for binomial data, χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;84.621). As for the cotyledons, the proportion of bleached cotyledons drastically increased between [Km]=50mg/L and [Km]=100mg/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, golden line; logistic generalised linear model for binomial data, χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;655.1). When [Km]\u0026le;50mg/L, less than 10% of germinated seedlings had bleached cotyledons; when [Km]\u0026ge;100mg/L, more than 85% of germinated seedlings had bleached cotyledons. Besides, cotyledon size was also significantly smaller for [Km]\u0026ge;100mg/L compared to the [Km]\u0026thinsp;=\u0026thinsp;0 and 50 mg/L groups, while the [Km]=50mg/L group also had significantly smaller cotyledons compared to the [Km]=0mg/L group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, df\u0026thinsp;=\u0026thinsp;5, one-way nested ANOVA using linear mixed models).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese measurements show that [Km]=100mg/L was the lowest concentration required for most negative effects on seeds to show a clear cut off compared to [Km]=50mg/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Therefore, I set [Km]=100mg/L as the threshold concentration to inhibit seed growth.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2-Direct somatic embryogenesis\u003c/h2\u003e \u003cp\u003eDifferent tissue types differ greatly in the ability to produce direct somatic embryos under their own 6-benzylaminopurine concentration ([BAP]) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, binomial generalised linear model, χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;58.30, df\u0026thinsp;=\u0026thinsp;3). Shoot apical meristems (SAM) were significantly better at producing direct somatic embryos than other types of explants: nearly 80% of all SAM explants produced embryos after seven weeks in the light period (i.e. 4 weeks after finishing the kanamycin resistance test) and each explant produced from 2\u0026thinsp;~\u0026thinsp;50 embryos/explant (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u0026amp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Some hypocotyls were also good at producing the embryos, but only\u0026thinsp;~\u0026thinsp;10% of all hypocotyls could produce embryos ranging from 5\u0026thinsp;~\u0026thinsp;50 embryos/explant (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u0026amp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Leaves were the worst at producing the embryos: only 1 leaf from three-week-old seedlings produced 1 tiny embryo (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec) and another leaf from six-week-old young plants produced two tiny embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). The embryos from SAMs were also best at rooting: more than 60% embryos cut from the original SAMs managed to produce roots \u0026ge;1cm in the rooting media, while only about 30% of embryos from the hypocotyls managed to do so and none of the leaf explants rooted (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). When the embryos (regardless of where the embryos come from) successfully rooted in the rooting media (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef), more than 80% of them survived the whole acclimatising process and grew healthily in the glasshouse (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg-h).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDifferent tissue types\u0026rsquo; ability for producing direct somatic embryos and their embryos\u0026rsquo; rooting and surviving status during acclimitising process.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTissue type (include plate number)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of explants that produced direct somatic embryos (n out of 14/plate)*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNumber of embryos per explant that produced direct somatic embryos (range in some cases)*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal number of embryos cut out from each type of explant\u0026dagger;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal number of embryos from each tissue type that managed to root in rooting media\u0026dagger;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTotal number of embryos from each tissue type that survived the whole acclimatising process\u0026dagger;\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypocotyl 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026ndash;50/explant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e~\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypocotyl 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrue leaves from 3-week-old seedlings 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrue leaves from 3-week-old seedlings 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrue leaves from 6-week-old young plants 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrue leaves from 6-week-old young plants 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShoot apical meristem 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u0026ndash;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e~\u0026thinsp;260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e132\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShoot apical meristem 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u0026ndash;50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e*These data were recorded 4 weeks after finishing kanamycin resistance test\u0026thinsp;=\u0026thinsp;7 weeks into the light period.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u0026dagger;These data were recorded 16 weeks after finishing kanamycin resistance test, when this direct somatic embryogenesis experiment finished.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDuring this experiment, I noticed that the very base of some true leaves were left on the shoot apical meristems (SAM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), as they were extremely hard to remove at the beginning of the kanamycin resistance test when the shoot of the seedling was only ~1cm tall. Therefore, these \u0026lsquo;leftovers\u0026rsquo; of the true leaves grew at [BAP]=1mg/L. The \u0026lsquo;leftovers\u0026rsquo; later grew bigger along with the main part of the SAMs and produced some embryos from the original cutting site (~\u0026thinsp;15\u0026ndash;20 embryos in total out of ~\u0026thinsp;5 \u0026lsquo;leftovers\u0026rsquo; I identified). Interestingly, both the \u0026lsquo;leftover\u0026rsquo; leaves and their embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee) seemed to survive much better than the leaves and the leaf-produced embryos that grew on [BAP]\u0026thinsp;=\u0026thinsp;1.5mg/L medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec-d). ~10 of them even successfully developed into mature plants. I wondered whether the leaves may be healthier and survive better if I reduced the [BAP] to 1mg/L (i.e. same concentration as culturing hypocotyls and shoot apical meristems). Therefore, in a small follow-up experiment, I changed the BAP concentration to 1mg/L for all tissue types for direct somatic embryogenesis while other conditions remained the same. As was expected, the ability of direct somatic embryogenesis was greatly improved for true leaves from 3-week old seedlings\u0026mdash;2 out of 16 explants (vs. original 2 out of 56) produced direct somatic embryos and the number of embryos per explant ranged from 5\u0026ndash;50 (vs. original 1\u0026ndash;2). Besides, if regenerated plantlets are transferred into a larger container with resupplied rooting medium, the plantlets can flower and set seeds \u003cem\u003ein vitro\u003c/em\u003e in 3\u0026ndash;6 weeks after being separated for rooting.\u003c/p\u003e \u003cp\u003eThese results show that hypocotyl, leaf and shoot apical meristem were all capable of producing direct somatic embryos despite huge differences. To maximise the efficiency and reduce total time of somatic embryogenesis, it would be better to use all materials from healthy 3-week-old seedlings (i.e. hypocotyl, cotyledon, true leaf and shoot apical meristem).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e4.1-Great potential for direct somatic embryogenesis in \u003cem\u003ePortulaca amilis\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003ePortulaca amilis\u003c/em\u003e showed great potential in producing direct somatic embryos and it is relatively easy to induce direct somatic embryos and grow the embryos into mature plants (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). The whole regeneration process did not encounter the common bottlenecks that constrain somatic embryogenesis in other species, such as hard to induce direct somatic embryos, complex hormone combination, low survival of regenerated plantlets, long waiting time in various regeneration process (Litz and Gray, \u003cspan class=\"CitationRef\"\u003e1995\u003c/span\u003e; Park et al., \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e; Suzuki et al., \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e; Safdari and Kazemitabar \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e; Juturu et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). First, it is easy to propagate large numbers of sterile seedlings \u003cem\u003ein vitro\u003c/em\u003e\u0026mdash;the seeds are tiny (~\u0026thinsp;0.5mm in diameter, Judd and Wunderlin, \u003cspan class=\"CitationRef\"\u003e1981\u003c/span\u003e) and can be thoroughly sterilised in large batches within 45 minutes using less hazardous reagents. Larger seeds (\u0026ge;\u0026thinsp;1mm, e.g. \u003cem\u003ePortulaca oleracea\u003c/em\u003e) usually require higher concentration of NaClO solution (~\u0026thinsp;1.5\u0026ndash;2.5% (m/m)) and some even require HgCl\u003csub\u003e2\u003c/sub\u003e, which are both more hazardous to both people and seeds (e.g. Bagheri et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e; Raut et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sedaghati et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Second, it only takes 3 weeks for the sterile seedlings to be ready as explant sources for direct somatic embryogenesis. These explants need no extra sterilisation compared to explants from non-sterile plants. For example, stem and leaf cuttings from non-sterile plants often need to be washed, sterilised with either 70\u0026thinsp;~\u0026thinsp;75% ethanol, ~\u0026thinsp;1% NaClO or 0.1% HgCl\u003csub\u003e2\u003c/sub\u003e and properly dried in the flow hood (e.g. \u003cem\u003ePortulaca grandiflora\u003c/em\u003e and \u003cem\u003eP. pilosa\u003c/em\u003e, Safdari and Kazemitabar \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e; Chen et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Third, the hormone combinations for inducing large numbers of direct somatic embryos in \u003cem\u003eP. amilis\u003c/em\u003e is relatively simple compared to its relatives such as \u003cem\u003eP. oleracea\u003c/em\u003e, \u003cem\u003eP. pilosa\u003c/em\u003e and \u003cem\u003eP. grandiflora\u003c/em\u003e. (Safdari and Kazemitabar \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e; Sedaghati et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Chen et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). In \u003cem\u003eP. amilis\u003c/em\u003e, 1mg/L of 6-benzylaminopurine (BAP) is sufficient to induce large numbers of direct somatic embryos from hypocotyl, leaves and shoot apical meristems (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e)\u0026mdash;all parts of the shoot in a three-week-old seedling can produce direct somatic embryos, which is not common in other species. For \u003cem\u003eP. oleracea\u003c/em\u003e, stems cannot produce direct somatic embryos and must go through the callus phase, while leaves need both BAP and 1-naphthaleneacetic acid (NAA) under a certain combination to produce large numbers of direct somatic embryos (Sedaghati et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). For \u003cem\u003eP. pilosa\u003c/em\u003e, the leaves need both thidiazuron (TDZ) and NAA to produce direct somatic embryos (Chen et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). For \u003cem\u003eP. grandiflora\u003c/em\u003e, the leaves cannot produce direct somatic embryos and require both BAP and NAA to induce somatic embryos from callus (Safdari and Kazemitabar \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). Lastly, when the embryos of \u003cem\u003eP. amilis\u003c/em\u003e successfully rooted in the rooting medium (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ef), one can acclimatise them easily in large batches in a growth cabinet with \u0026gt;\u0026thinsp;80% survival and hardly any abnormal development, as long as the soil is moist and well-drained (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThese four advantages in direct somatic embryogenesis, plus the advantages of being a diploid and having a fully sequenced genome, make \u003cem\u003ePortulaca amilis\u003c/em\u003e a good model species for future studies, including transformation (Danin et al., \u003cspan class=\"CitationRef\"\u003e1978\u003c/span\u003e; Judd and Wunderlin, \u003cspan class=\"CitationRef\"\u003e1981\u003c/span\u003e; Walter et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Gilman et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). To facilitate future studies involving direct somatic embryogenesis of \u003cem\u003eP. amilis\u003c/em\u003e, I hereby provide an optimised protocol based on result section \u003cspan class=\"InternalRef\"\u003e3.2\u003c/span\u003e (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), which is simple, efficient and economical for batch propagation or maintain large quantity of sterile plant lines. The main procedure include (1) sterilising seeds; (2) plate and grow seeds into 3-week-old seedlings; (3) cut the seedlings into hypocotyl, cotyledon, true leaves and shoot apical meristem; (4) plate and culture in dark period (range from 1-maximum 3 weeks); (5) culture in light period to induce direct somatic embryos and grow them into \u0026ge;5mm mini plantlets (range from 4\u0026ndash;8 weeks); (6) separate the mini plantlets for rooting (1\u0026ndash;2 weeks); (7.1) keep rooted plantlets growing in sterile environment to flower and set seeds (2\u0026ndash;4 weeks), or (7.2) acclimatise rooted plantlets in sterile water (3\u0026ndash;4 days) and then sterile soil (7\u0026ndash;12 days) in growth chamber; (8) acclimatise rooted plantlets in non-sterile soil in the glasshouse (3\u0026ndash;4 weeks to obtain full-sized plants setting seeds). The whole process from sterilising seeds to regenerated mature plants setting seeds (sterile/non-sterile) takes only 3\u0026ndash;4 months, while from explants to moving plantlets to soil already takes nearly 2 months in \u003cem\u003eP. oleracea\u003c/em\u003e (Sedaghati et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). Besides, the acclimatisation setting in step 6\u0026ndash;7 can be reused and takes little space due to smaller plant size, thus highly economical.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eOptimised protocol for direct somatic embryogenesis.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eprocedure\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003emedium/soil condition\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003egrowth/treatment condition\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003etotal timing\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1. sterile seeds\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(1)70% ethanol: 1 min;\u003c/p\u003e\n \u003cp\u003e(2)1 Milton mini tablet sterilised water: 25 min;\u003c/p\u003e\n \u003cp\u003e(3)sterile water: 3 times, 1min/time\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esterile environment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30-40min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2. plate and grow seeds into 3-week-old seedlings\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003efull strength MS with Gamborg B5 vitamin;\u003c/p\u003e\n \u003cp\u003e3% (m/v) sucrose;\u003c/p\u003e\n \u003cp\u003e0.7% (m/v) bacto-agar;\u003c/p\u003e\n \u003cp\u003epH\u0026thinsp;=\u0026thinsp;5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etemperature: 25\u0026deg;C;\u003c/p\u003e\n \u003cp\u003elight intensity: 5300 Lux;\u003c/p\u003e\n \u003cp\u003ephotoperiod: 16h light/8h dark;\u003c/p\u003e\n \u003cp\u003esterile environment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 weeks\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3. cut the seedlings into hypocotyl, cotyledons, true leaves and shoot apical meristem\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecut on thin layer of sterile water to avoid dehydration\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esterile environment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u0026ndash;2 hours depending on quantity\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4. plate and culture explants in darkness\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003efull strength MS with Gamborg B5 vitamin;\u003c/p\u003e\n \u003cp\u003e3% (m/v) sucrose;\u003c/p\u003e\n \u003cp\u003e0.7% (m/v) bacto-agar;\u003c/p\u003e\n \u003cp\u003e1mg/L BAP;\u003c/p\u003e\n \u003cp\u003epH\u0026thinsp;=\u0026thinsp;5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etemperature: 25\u0026deg;C;\u003c/p\u003e\n \u003cp\u003elight intensity: 0 Lux (complete darkness)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1-maximum 3 weeks\u0026dagger;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5. plate and culture explants in light to induce direct somatic embryos and grow them into \u0026ge;5mm plantlets\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003efull strength MS with Gamborg B5 vitamin;\u003c/p\u003e\n \u003cp\u003e3% (m/v) sucrose;\u003c/p\u003e\n \u003cp\u003e0.7% (m/v) bacto-agar;\u003c/p\u003e\n \u003cp\u003e1mg/L BAP;\u003c/p\u003e\n \u003cp\u003epH\u0026thinsp;=\u0026thinsp;5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05*;\u003c/p\u003e\n \u003cp\u003erefresh media once every 2\u0026ndash;3 weeks to keep inducing embryos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etemperature: 25\u0026deg;C;\u003c/p\u003e\n \u003cp\u003elight intensity: 5300 Lux;\u003c/p\u003e\n \u003cp\u003ephotoperiod: 16h light/8h dark;\u003c/p\u003e\n \u003cp\u003esterile environment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u0026ndash;8 weeks\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6. cut and root regenerated plantlets until roots reach 1-2cm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003efull strength MS with Gamborg B5 vitamin;\u003c/p\u003e\n \u003cp\u003e3% (m/v) sucrose;\u003c/p\u003e\n \u003cp\u003e0.7% (m/v) bacto-agar;\u003c/p\u003e\n \u003cp\u003epH\u0026thinsp;=\u0026thinsp;5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etemperature: 25\u0026deg;C;\u003c/p\u003e\n \u003cp\u003elight intensity: 5300 Lux;\u003c/p\u003e\n \u003cp\u003ephotoperiod: 16h light/8h dark;\u003c/p\u003e\n \u003cp\u003esterile environment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u0026ndash;2 weeks\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.1 keep growing in sterile environment until fruiting\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003efull strength MS with Gamborg B5 vitamin;\u003c/p\u003e\n \u003cp\u003e3% (m/v) sucrose;\u003c/p\u003e\n \u003cp\u003e0.7% (m/v) bacto-agar;\u003c/p\u003e\n \u003cp\u003epH\u0026thinsp;=\u0026thinsp;5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05*;\u003c/p\u003e\n \u003cp\u003erefresh media once every 2\u0026ndash;3 weeks to keep plantlets growing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etemperature: 25\u0026deg;C;\u003c/p\u003e\n \u003cp\u003elight intensity: 5300 Lux;\u003c/p\u003e\n \u003cp\u003ephotoperiod: 16h light/8h dark;\u003c/p\u003e\n \u003cp\u003esterile environment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u0026ndash;4 weeks\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.2 acclimatise rooted plantlets in sterile water, then sterile soil in growth chamber\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(1) sterile water in 2ml centrifuge tubes: 3\u0026ndash;4 days, refill with water if water level is low;\u003c/p\u003e\n \u003cp\u003e(2) sterile soil in small pots (M3 soil:sand:perlite\u0026thinsp;=\u0026thinsp;2:2:1): 7\u0026ndash;12 days, water once a day or once every two days based on soil moisture\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etemperature: 25\u0026deg;C;\u003c/p\u003e\n \u003cp\u003elight intensity: 5300 Lux;\u003c/p\u003e\n \u003cp\u003ephotoperiod: 16h light/8h dark;\u003c/p\u003e\n \u003cp\u003esterile to non-sterile environment;\u003c/p\u003e\n \u003cp\u003ecentrifuge tubes and small pots kept in 1000\u0026micro;l pipette tip boxes with lids gradually opening\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u0026ndash;16 days\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8. acclimatise plantlets in glasshouse (after 7.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enon-sterile soil in medium-large pots (0.2-0.5L);\u003c/p\u003e\n \u003cp\u003eM3 soil: sand: perlite\u0026thinsp;=\u0026thinsp;2:2:1;\u003c/p\u003e\n \u003cp\u003eonce a day or once every two days based on soil moisture\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etemperature: 18\u0026ndash;25\u0026deg;C;\u003c/p\u003e\n \u003cp\u003elight intensity: ~5300 Lux, vary a bit based on temperature;\u003c/p\u003e\n \u003cp\u003ephotoperiod: 16h light/8h dark;\u003c/p\u003e\n \u003cp\u003enon-sterile environment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u0026ndash;4 weeks for full sized plants setting seeds\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e*adjusted by 0.5mol/L KOH and 0.5mol/L HCl\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e\u0026dagger;1 week is sufficient to induce direct somatic embryos based on a small follow-up experiment. Depending on the purpose, dark period can be longer, but no longer than 3 weeks due to reduced explant survival rate.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e4.2-Threshold kanamycin (Km) concentration for \u003cem\u003ePortulaca amilis\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eThe threshold kanamycin concentration ([Km]) for \u003cem\u003ePortulaca amilis\u003c/em\u003e is 50mg/L for tissues and 100mg/L for seeds, much lower than the threshold [Km] for tissues and seeds of \u003cem\u003ePortulaca oleracea\u003c/em\u003e (both 250mg/L, Sedaghati et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Despite from the same genus, two species respond to Km differ greatly. For the four tissue types of \u003cem\u003eP. amilis\u003c/em\u003e I used, all regeneration events, from callus to direct somatic embryos, completely disappeared when [Km] was only 50mg/L (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb-c). But for \u003cem\u003eP. oleracea\u003c/em\u003e, regeneration only stopped when [Km] reached 250mg/L for leaves\u0026mdash;five times the threshold [Km] of \u003cem\u003eP. amilis\u003c/em\u003e (Sedaghati et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). For the seeds, germination was not inhibited by increasing [Km] in \u003cem\u003eP. amilis\u003c/em\u003e\u0026mdash;more than 90% of all seeds still germinated normally on [Km]=250mg/L (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea, light green line). I therefore used several other parameters (e.g. the proportion of germinated seeds that produce true leaves) to comprehensively determine threshold [Km] (see Result 3.1.1). In \u003cem\u003eP. oleracea\u003c/em\u003e, however, germination rate decreased with increasing [Km] and no seeds germinated at 250mg/L, making the threshold [Km] obvious (Sedaghati et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe tissues of \u003cem\u003ePortulaca amilis\u003c/em\u003e are extremely sensitive to Km while the seeds are less so compared to other species. Past studies show that the threshold [Km] for leaf or stem explants ranges from 15mg/L to 250mg/L, above which either regeneration stops completely or the explants all died (Filatti et al., 1987; Bagheri et al, \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e; Bahmanker et al., 2015, Sedaghati et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). While the tissues of \u003cem\u003eP. amilis\u003c/em\u003e stay near the lower boundary of threshold [Km], the tissues of many species fall between 100mg/L-200mg/L, such as honeydews (\u003cem\u003eCucumis melo\u003c/em\u003e), pigeon peas (\u003cem\u003eCajanus cajan\u003c/em\u003e) and \u003cem\u003ePhysalis pruinosa\u003c/em\u003e (e.g. Ren et al., \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e; Raut et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Swartwood and Van Eck, \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). For seeds, the threshold [Km] ranges from 75mg/L to 250mg/L and seeds of \u003cem\u003eP. amilis\u003c/em\u003e lie in the middle (Gatzek et al., \u003cspan class=\"CitationRef\"\u003e2002\u003c/span\u003e; Darqui et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; Niazian et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sedaghati et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). These threshold [Km] were chosen either based on germination rate, if germination rate decreases with increasing [Km], or other criteria such as proportion of germinated seeds that produced side roots, if germination rate was not negatively affected by increasing [Km].\u003c/p\u003e\n \u003cp\u003eAs we can see, the threshold [Km] depends on the species and tissue/organ types and is highly variable even for closely related species (e.g. \u003cem\u003ePortulaca amilis vs. P. oleracea\u003c/em\u003e). Therefore, if one decides to use kanamycin as the selection reagent for transforming a new species, variant or tissue/organ type, they must perform a kanamycin resistance test ahead of time. Otherwise, a too low [Km] may cause \u0026lsquo;false positive\u0026rsquo; results (i.e. non-transformed plants survive/regenerate on selective media) while a too high [Km] may kill everything, no matter whether the tissues/organs are successfully transformed or not (e.g. Sedaghati et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e4.3-Outlook and conclusion\u003c/h2\u003e\n \u003cp\u003eDespite various advantages in direct somatic embryogenesis of \u003cem\u003ePortulaca amilis\u003c/em\u003e under the above experimental procedure, it is worthwhile investigate other aspects in the regeneration process that may be critical to future experimental setups. First, tissues from young seedlings are potentially good explant source, as actively growing tissues have more endogenous hormones and require less external ones for somatic embryogenesis (Deo et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kim et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e; Sedaghati et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). Apart from \u003cem\u003ePortulaca amilis\u003c/em\u003e, several other species use young tissues like hypocotyl, cotyledons, germinating seeds and immature zygotic embryos to produce somatic embryos (e.g. \u003cem\u003eGossypium\u003c/em\u003e, \u003cem\u003eSolanum lycopersicum\u003c/em\u003e, \u003cem\u003eZea mays\u003c/em\u003e, \u003cem\u003eArabidopsis\u003c/em\u003e; Fillatti et al., \u003cspan class=\"CitationRef\"\u003e1987\u003c/span\u003e; Su et al., \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e; Jurutu et al., 2015; Lowe et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ikeuchi et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). Second, a few stress factors can positively or negatively affect somatic embryogenesis. The two critical ones are wounding and antibiotic toxicity, which require particular attention as somatic embryogenesis is often involved in \u003cem\u003eAgrobacterium-\u003c/em\u003emediated transformation to knockout or overexpress genes of interest (Anami et al., \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ikeuchi et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). Wounding induces hormone synthesis and in general promotes somatic embrygenesis (Ikeuchi et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mozgov\u0026aacute; et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). On the other hand, various antibiotics often inhibit somatic embryogenesis even for the ones that do not kill cells like cefotaxime (i.e. the most commonly used antibiotics to remove \u003cem\u003eAgrobacterium\u003c/em\u003e after transformation; Ling et al., \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e; Teixeira da Salva and Fukai, 2001). In \u003cem\u003ePortualca amilis\u003c/em\u003e, my small pilot experiment (Zhao, \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e) showed that direct somatic embryogenesis was greatly inhibited when the tissues are exposed to moderate concentration of cefotaxime for only two hours (150mg/L, commonly used in transformation; Ling et al., \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e; Teixeira da Salva and Fukai, 2001; Sedaghati et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, although \u003cem\u003eP. amilis\u003c/em\u003e showed great direct somatic embryogenesis ability under benign conditions, one need to investigate whether such ability can be maintained in successfully transformed cells.\u003c/p\u003e\n \u003cp\u003eIn conclusion, this study demonstrated a simple, efficient way to batch propagate direct somatic embryos of \u003cem\u003ePortulaca amilis\u003c/em\u003e and also reported kanamycin resistance ability for tissue and seeds. The results can be applied to various future studies involving direct somatic embryogenesis, especially towards developing a successful transformation protocol in the species.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBAP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e6-benzylaminopurine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHCl\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehydrochloric acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKm\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ekanamycin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKOH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epotassium hydroxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMS medium\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMurashige and Skoog medium (MS)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements:\u003c/p\u003e\n\u003cp\u003eI would like to thank Cambridge International Trust and Newnham College for funding my PhD (tuition and stipend), part of which gave rise to this manuscript. I would like to thank the following persons and teams for providing me with technical and data analysis support, access to facilities and manuscript revision suggestions (in alphabetical order of last names and organisations): Dr. Julie Bailey, Dr. Sam Brockington, Professor Kate Fleet, Professor Beverley Glover, Fiona Holder, Professor Nik Cunniffe (special thanks for statistical analysis advice), Dr. Jiafu Tan (special thanks for tissue culture advice), Dr. Xiaoyu Wang; Cambridge University Botanic Garden horticultural team, Newnham College, Sainsbury Lab security, reception, administrative and technical staff.\u003c/p\u003e\n\u003cp\u003eStatements and Declarations:\u003c/p\u003e\n\u003cp\u003eThis work was supported by Cambridge International \u0026amp; Newnham College Scholarship funded by the Cambridge Commonwealth, European \u0026amp; International Trust. The author has no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003eConflict of interest:\u003c/p\u003e\n\u003cp\u003eThe author has no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003eAuthor contribution statement:\u003c/p\u003e\n\u003cp\u003eI am the only author for the manuscript, responsible for experimental design, data collection, analyses and interpretation, manuscript writing and publishing.\u003c/p\u003e\n\u003cp\u003eEthics declaration:\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eSupplementary information:\u003c/p\u003e\n\u003cp\u003eApart from data directly presented in the tables, all spreadsheets and R code (version 4.2.1, detailed comments included) used for data analyses in this manuscript are included in supplementary information as an .xlsx file (compiled raw data) and .rmd file (compiled R code) for running the code with the data in R.\u003c/p\u003e\n\u003cp\u003eData availability statement:\u003c/p\u003e\n\u003cp\u003eAll raw data and code included in supplementary information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAnami, S., Njuguna, E., Coussens, G., Aesaert, S. and Van Lijsebettens, M. 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Annals of Botany 57: 443-462.\u003c/li\u003e\n \u003cli\u003eXiong, Y., Chen, S., Wei, Z., Yu, X., Pang, J., Zhang, T., Wu, K., Ren, H., Jian, S., Teixeira da Silva, J.A., and Ma, G. (2021). In vitro flowering and fruiting in \u003cem\u003ePortulaca pilosa\u003c/em\u003e L. South African Journal of Botany 140: 1-3.\u003c/li\u003e\n \u003cli\u003eZhao, Y. (2025). Towards a systematic analysis and comparison of perianth evolution in Caryophyllineae in the context of the synflorescence [Apollo - University of Cambridge Repository]. https://doi.org/10.17863/CAM.122697\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Portulaca amilis, direct somatic embryogenesis, kanamycin resistance, tissue culture, Portulacaceae","lastPublishedDoi":"10.21203/rs.3.rs-8790648/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8790648/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlant somatic embryogenesis has critical applications in science, agriculture and industry. A successful somatic embryogenesis protocol requires optimisation on numerous factors such as growth regulators, tissue type, light, temperature, survival rate and can be species specific. Due to lack of callus phase, direct somatic embryogenesis is usually more efficient but can be recalcitrant to induce. In this study, I established an efficient, simple way to induce, regenerate and acclimatise direct somatic embryos from various young shoot tissues of three-week-old seedlings in \u003cem\u003ePortulaca amilis\u003c/em\u003e (Portulacaceae) with high survival rate. The regenerated plantlets can be maintained in sterile environment and set seeds \u003cem\u003ein vitro\u003c/em\u003e or acclimatised and set seeds in non-sterile environment, flexible for different purposes. The species has great potential as a model species for biochemical, physiological, medical, genetical and morphological studies. The only external hormone is 1mg/L 6-benzylaminopurine and it only takes three to four months from preparing explants from sterile seeds to mature regenerated plants setting seeds in non-sterile glasshouses. I also found out the threshold kanamycin concentration in both tissue (50mg/L) and seeds (100mg/L) for \u003cem\u003eP. amilis\u003c/em\u003e. Both direct somatic embryogenesis and kanamycin resistance will be informative for future genetic studies involving transformation.\u003c/p\u003e","manuscriptTitle":"A simple way to batch propagate direct somatic embryos and kanamycin resistance of Portulaca amilis (Portulacaceae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-17 07:44:02","doi":"10.21203/rs.3.rs-8790648/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0d7deee8-0038-4e73-bd69-204ef536b4a6","owner":[],"postedDate":"February 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-12T08:40:17+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-17 07:44:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8790648","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8790648","identity":"rs-8790648","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}