Bioreactor application on adventitious root culture of <Emphasis Type="Italic">Astragalus membranaceus</Emphasis>

Astragalus membranaceus is one of the most widely used traditional medicinal herbs in China, but the time required to generate a useful product in the field production is long. The growth of adventitious root cultures was compared between cultures grown in solid, liquid, or a 5-L balloon-type bubble bioreactor. The maximum growth ratio (final dry weight/initial dry weight) was determined for adventitious roots grown in the bioreactor. Studies carried out to optimize biomass production of adventitious roots compared adventitious root growth from various inoculum root lengths, inoculum densities, and aeration volume in the bioreactors. The maximum growth ratio occurred in treatments with a 1.5-cm inoculum root length, with 30 g (fresh weight) of inoculum per bioreactor or with an aeration volume of 0.1 vvm (air volume/culture medium volume per min). The polysaccharide, saponin, and flavonoid content of roots from bioreactor-grown cultures were compared to roots from field-grown plants grown for 1 and 3 yr. Total polysaccharide content of adventitious roots in the bioreactor (30.0 mg g⁻¹ dry weight (DW)) was higher than the roots of 1-yr-old (13.8 mg g⁻¹ DW) and 3-yr-old (21.1 mg g⁻¹ DW) plants in the field. Total saponin (3.4 mg g⁻¹ DW) and flavonoid (6.4 mg g⁻¹ DW) contents were nearly identical to 3-yr-old roots and higher than that of 1-yr-old roots under field cultivation.     

Cryopreservation of <Emphasis Type="Italic">Panax ginseng</Emphasis> Adventitious Roots

We tested desiccation and/or vitrification procedures to cryopreserve the adventitious roots of Panax ginseng, the source of commercially produced ginsenosides. When only desiccation was applied, the post-freeze survival of 3- to 4-mm root tips was <14% regardless of the composition of the preculture medium or the explant origin. Callus formation was frequently observed after cryopreservation. In contrast, 90% survival and 32.5% root formation efficiency were achieved after cryopreservation when a vitrification protocol was followed. Adventitious root cultures in flasks and bioreactors were reestablished from root tips cryopreserved by vitrification. A prolonged lag-phase and lower biomass production were recorded in post-freeze-regenerated cultures compared with control roots that were subcultured four times in flasks. However, biomass accumulations did not differ between control and regenerated roots at the end of the sixth subculturing period. After 40 days of culture in bioreactors, a mean value of 12.5 g dw L⁻¹ was recorded for post-freeze-regenerated cultures versus 9.1 g dw L⁻¹ for the control roots. Production of triol and diol ginsenosides in our bioreactor cultures also was enhanced after cryopreservation, by 41.0% and 89.8%, respectively. These results suggest that the vitrification method is successful for cryopreservation of P. ginseng adventitious roots.     

Production of Biomass and Ginsenosides from Adventitious Roots of Panax ginseng in Bioreactor Cultures

Organic nutrients play a central role during Panax ginseng adventitious root culture in bioreactor systems. To understand how the nutrient elements were uptaken during the adventitious root growth as well as the production of biomass and natural ginsenosides, a biotechnological approach to identifying the nutritional physiology of ginseng in a commercial‐scale bioreactor was necessary. Normal MS medium nutrient in the bioreactor culture of adventitious roots resulted in slow growth, low biomass, and Rg and Rb ginsenoside contents. When the ginsenoside production increased to higher levels, a group of regulatory nutritional elements that have the potential to interact with biomass was identified. The effects of the salt strength of the medium, of macroelements, metal elements, the ammonia/nitrate ratio, sucrose concentration, and osmotic agents on the growth, the formation of biomass and the production of ginsenosides from adventitious roots were investigated. Appropriate conditions allowed for a maximum ginsenoide production of up to 12.42 [mg/g DW] to be obtained after 5 weeks of culture. The results demonstrated that the key organic nutrients can be regulated to improve the biomass and growth, and increase the ginsenoside yield in bioreactor cultures of P. ginseng adventitious roots.     

Temporal changes in the growth, saponin content and antioxidant defense in the adventitious roots of Panax ginseng subjected to nitric oxide elicitation

Nitric oxide (NO) is a diffusible, gaseous signaling molecule. In plants, NO influences growth and development, and it can also affect plant responses to various stresses. Because NO induces root differentiation and interacts with reactive oxygen species, we examined the temporal effect of NO elicitation on root growth, saponin accumulation and antioxidant defense responses in the adventitious roots of mountain ginseng (Panax ginseng). The observations revealed that NO is involved in root growth and saponin production. Elicitation with sodium nitroprusside (SNP) activated O₂ ⁻-generating NADPH oxidase (NOX) activity, which most probably subsequently enhanced growth of adventitious roots of mountain ginseng. A severe inhibition of NOX activity and decline in dry weight of SNP elicited adventitious roots in the presence of NOX inhibitor (diphenyl iodonium, DPI), which further supports involvement of NOX in root growth. Enhanced activities of antioxidant enzymes by SNP appear to be responsible for low H₂O₂, less lipid peroxidation, and modulation of ascorbate and non-protein thiol statuses in the adventitious roots of mountain ginseng. Dry mass, saponin content and NOX activity was related with NO content present in adventitious roots of mountain ginseng.     

Manipulation of ginsenoside heterogeneity of <Emphasis Type="Italic">Panax notoginseng</Emphasis> cells in flask and bioreactor cultivations with addition of phenobarbital

Structure-similar ginsenosides have different or even totally opposite biological activities, and manipulation of ginsenoside heterogeneity is interesting and significant to biotechnological application. In this work, addition of 1 mM phenobarbital to cell cultures of Panax notoginseng at a relatively high inoculation size of 7.6 g dry cell weight (DW)/L enhanced the production of protopanaxatriol-type (Rg₁ + Re) ginsenosides in both shake flask and airlift bioreactor (ALR, 1 L working volume). The content of Rg₁ + Re in the ALR was increased from 42.5 ± 4.0 mg per gram DW in untreated cell cultures (control) to 56.4 ± 4.6 mg per gram DW with addition of 1.0 mM phenobarbital. The maximum productivity of Rg₁ + Re in the ALR reached 5.66 ± 0.38 mg L⁻¹ d⁻¹, which was almost 3.3-fold that of control. The maximum ratio of the detectable ginsenosides protopanaxatriol:protopanaxadiol (Rb₁) was 7.6, which was about twofold that of control. The response of protopanaxadiol 6-hydroxylase (P6H) activity to phenobarbital addition coincided with the above-mentioned change of ginsenoside heterogeneity (distribution). Phenobarbital addition is considered as a useful strategy for manipulating the ginsenoside heterogeneity in bioreactor with enhanced biosynthesis of protopanaxatriol by P. notoginseng cells.     

Influence of medium salt strength and nitrogen source on biomass and metabolite accumulation in adventitious root cultures of Pseudostellaria heterophylla

We investigated the effects of medium salt strength and ammonia/nitrate ratio on biomass production and metabolites accumulation of adventitious root. The medium with full-strength Murashige and Skoog (MS) reached the highest growth rate (16.77), and the contents of saponin and polysaccharide reached the peak (i.e., 0.65 and 24.85 %) at 3/4 MS and 1 MS, respectively. In case of ammonia/nitrate ratio, a NH₄ ⁺/NO₃ ^? ratio of 20:40 was optimal for the production of biomass and polysaccharide (23.27 %). In contrast, the content of saponin achieved the optimum (0.74 %) at a NH₄ ⁺/NO₃ ^? ratio of 30:30. In 5-L balloon-type bubble bioreactor (BTBB) cultures, an approximately 23-fold increase in biomass was recorded. The fresh weight (FW) and dry weight (DW) were 72.78 and 6.79 g per bioreactor with the contents of saponin (0.62 %) and polysaccharide (17.32 %), respectively. It indicated potential application to produce adventitious roots of pseudostellaria heterophylla with bioreactors on large scale in commercial.     

Dynamic change of metabolites and nutrients in suspension cells of <Emphasis Type="Italic">Panax Quinquefolium</Emphasis> L. in bioreactor

In order to understand how the nutrient elements were taken up during the cell growth as well as the production of metabolites, it was quite necessary to identify the dynamic change of metabolites and nutrients in suspension cells of Panax quinquefolium in bioreactor. In this study, dynamic accumulation of biomass and ginsenosides Re, Rb1 and polysaccharide as well as major nutrients consumption in cell suspension culture of P. quinquefolium in a 5-L stirred tank bioreactor were investigated. The dry cell weight and the contents of ginsenosides Re, Rb1 and polysaccharide reached the maximum peak simultaneously on about 21 days and the results showed that cell growth and metabolites synthesis related to nutrients consumption. For this reason, we supposed that the contents of metabolites can be increased through added nutrient at the right moment. These results provided theory reference for two-stage or continuous perfusion culture in suspension cells of P. quinquefolium in bioreactor.     

Influence of mist intervals and aeration rate on growth and second metabolite production of Pseudostellaria heterophylla adventitious roots in a siphon-mist bioreactor

Plant adventitious root culture in bioreactors is a promising alternative for the efficient production of medicinal herbs. Adventitious roots of Pseudostellaria heterophylla were induced from callus and then cultivated in a siphon-mist bioreactor. An orthogonal test established that the optimal medium for adventitious root induction was MS medium supplemented with 1.0 mg/L naphthaleneacetic acid and 2.0 mg/L 3-indolybutyric acid. Under these conditions, the average root number was more than 14 on each 1.0 cm diameter callus and the rooting rate reached 100%. The bioreactor was equipped with an integral siphon-spraying device designed to automatically supply the liquid medium. The operation parameters of the bioreactor were assessed by varying the mist interval and the aeration velocity. The mist interval was negatively related to average growth rate of the adventitious roots and positively related to saponin and polysaccharide content. A relatively high aeration rate was necessary to achieve the maximum biomass production, but the secondary metabolite production was not enhanced by increasing the aeration velocity.     

Highly efficient production of diverse rare ginsenosides using combinatorial biotechnology

The rare ginsenosides are recognized as the functionalized molecules after the oral administration of Panax ginseng and its products. The sources of rare ginsenosides are extremely limited because of low ginsenoside contents in wild plants, hindering their application in functional foods and drugs. We developed an effective combinatorial biotechnology approach including tissue culture, immobilization, and hydrolyzation methods. Rh2 and nine other rare ginsenosides were produced by methyl jasmonate-induced culture of adventitious roots in a 10 L bioreactor associated with enzymatic hydrolysis using six β-glycosidases and their combination with yields ranging from 5.54 to 32.66 mg L⁻¹. The yield of Rh2 was furthermore increased by 7% by using immobilized BglPm and Bgp1 in optimized pH and temperature conditions, with the highest yield reaching 51.17 mg L⁻¹ (17.06% of protopanaxadiol-type ginsenosides mixture). Our combinatorial biotechnology method provides a highly efficient approach to acquiring diverse rare ginsenosides, replacing direct extraction from Panax plants, and can also be used to supplement yeast cell factories.     

Waterlogging tolerance in the tribe Triticeae: the adventitious roots of Critesion marinum have a relatively high porosity and a barrier to radial oxygen loss

Nine species from the tribe Triticeae – three crop, three pasture and three ‘wild’ wetland species – were evaluated for tolerance to growth in stagnant deoxygenated nutrient solution and also for traits that enhance longitudinal O₂ movement within the roots. Critesion marinum (syn. Hordeum marinum) was the only species evaluated that had a strong barrier to radial O₂ loss (ROL) in the basal regions of its adventitious roots. Barriers to ROL have previously been documented in roots of several wetland species, although not in any close relatives of dryland crop species. Moreover, the porosity in adventitious roots of C. marinum was relatively high: 14% and 25% in plants grown in aerated and stagnant solutions, respectively. The porosity of C. marinum roots in the aerated solution was 1·8–5·4‐fold greater, and in the stagnant solution 1·2–2·8‐fold greater, than in the eight other species when grown under the same conditions. These traits presumably contributed to C. marinum having a 1·4–3 times greater adventitious root length than the other species when grown in deoxygenated stagnant nutrient solution or in waterlogged soil. The length of the adventitious roots and ROL profiles of C. marinum grown in waterlogged soil were comparable to those of the extremely waterlogging‐tolerant species Echinochloa crus‐galli L. (P. Beauv.). The superior tolerance of C. marinum, as compared to Hordeum vulgare (the closest cultivated relative), was confirmed in pots of soil waterlogged for 21 d; H. vulgare suffered severe reductions in shoot and adventitious root dry mass (81% and 67%, respectively), whereas C. marinum shoot mass was only reduced by 38% and adventitious root mass was not affected.     

Effect of polyploidy induction on biomass and ginsenoside accumulations in adventitious roots of ginseng

Adventitious roots ofPanax ginseng C.A. Meyer (a natural tetraploid) were treated with 50 or 100 mg L^-1 colchicine for 12, 24,36, 48, or 60 h to induce polyploid (octoploid) roots. The largest number of octoploid roots was obtained with a 100 mg L^-1 colchicine treatment over 60 h. To verify that ginsenoside was being accumulated in the developing tissues, the tetraploid (control) and octoploid roots were cultured for 40 d in Murashige and Skoog media that lacked NH₄NO₃ but was supplemented with 2 mg L^-1 naphthaleneacetic acid and 50 g L_-1 sucrose. Levels of fresh and dry biomass were greater in the octoploid roots. Although total ginsenoside and Rb-group ginsenoside contents were less in the octoploid roots than in the tetraploids, the former had a higher amount of Rg-group ginsenosides (especially Rg1). These results demonstrate the benefit that polyploid adventitious roots provide in enhancing the production of secondary metabolites in ginseng.     

Production of saponions and polysaccharide in the presence of lactoalbumin hydrolysate in <Emphasis Type="Italic">Panax quinquefolium</Emphasis> L. cells cultures

In this article, ginsenosides and polysaccharide contents in suspension cells and native roots of Panax quinquefolium L. were studied. In order to enhance the contents of ginsenosides and polysaccharide in P. quinquefolium suspension cells, we tested the effects of lactoalbumin hydrolysate on the growth of P. quinquefolium suspension cell, synthesis of ginsenosides and polysaccharide in flask and bioreactor. In flask culture, cells growth ratio was significantly enhanced by the addition of lower concentration of lactoalbumin hydrolysate. Addition of 100 mg L⁻¹ lactoalbumin hydrolysate significantly enhanced the contents of total saponins (5.44 mg g⁻¹ DW) and the contents were 3.89-fold over the control group. Addition of lactoalbumin hydrolysate significantly promoted the accumulation of polysaccharide, except 200 mg L⁻¹ lactoalbumin hydrolysate. The highest total saponins yield (36.72 mg L⁻¹ DW) and polysaccharide yield (0.83 g L⁻¹ DW) were obtained at 100 mg L⁻¹ lactoalbumin hydrolysate. In a 5-L stirred tank bioreactor, the highest contents of total saponins and TRb group ginsenosides were achieved on day 26, while the effect of lactoalbumin hydrolysate on the contents of TRg group ginsenosides were insignificant. This result suggests that lactoalbumin hydrolysate might have triggered the enzyme activities for the synthesis of TRb group ginsenosides. Overall, the highest total saponins yield (31.37 mg L⁻¹ DW) and polysaccharide yield (1.618 g L⁻¹ DW) were obtained on day 26 and day 24 respectively and the polysaccharide yield was 1.95-fold higher than the shake flask culture (0.83 g L⁻¹ DW). These results provided theoretical reference for two-stage culture in suspension cells of P. quinquefolium in bioreactor.     

Nitrogen and phosphorus as the factors affecting ginsenoside production in hairy root cultures of <Emphasis Type="Italic">Panax quinquefolium</Emphasis> cultivated in shake flasks and nutrient sprinkle bioreactor

The study assesses the influence of different concentrations of nitrogen and phosphorus sources on ginsenoside biosynthesis in Panax quinquefolium hairy roots cultivated in shake flasks and a nutrient sprinkle bioreactor. The saponin content was determined using HPLC. The maximum yield (12.45 mg g^?1 dw) of the sum of six examined ginsenosides (Rb1, Rb2, Rc, Rd, Re and Rg1) in hairy roots cultivated in shake flasks was achieved in modified Gamborg B-5 medium containing 0.83 mM l^?1 phosphate, 12.4 mM l^?1 nitrate and 0.5 mM l^?1 ammonium. The yield itself was 1.93 times higher than that achieved in standard Gamborg medium. The modified medium also favourably influenced the biosynthesis of studied saponins in bioreactor cultures. The saponin content (35.11 mg g^?1 d.w.) was 2.75-times higher than that achieved in control medium.     

Improvement of ginsenoside production by <Emphasis Type="Italic">Panax ginseng</Emphasis> adventitious roots induced by γ-irradiation

In order to evaluate effects of γ-rays on adventitious root formation and ginsenoside production, embryogenic calli induced from cotyledon explants of Panax ginseng C.A. Meyer were treated with γ-rays of 0, 10, 30, 50, 70, and 100 Gy. The highest frequency of adventitious root formation of 75 % occurred at γ-irradiation of 30 Gy, which is considered adequate dosage for selecting mutant cell lines. Five mutated adventitious roots (MAR)3-lines out of the propagation of 142 adventitious root lines treated with 30 Gy were selected based a 100-fold increase in proliferation rate compared to control adventitious roots (CAR) and content of the seven major ginsenosides (Rb₁, Rb₂, R_c, R_d, R_e, R_f, and Rg₁) was determined. In the CAR and four of the MAR3-lines (except for MAR3-109), the Rb/Rg ratio was greater than 1.0, thereby indicating altered ginsenoside composition in these root lines. The HPLC analysis of the MAR3-13 and MAR3-26 lines confirmed different ginsenoside profiles, including the three unidentified ginsenoside candidates, Gm₁, Gm₂, and Gm₃. The ginsenosides of the MAR3-13 and MAR3-26 lines showed high hydroxyl and superoxide radical scavenging activities.     

Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate

Adventitious roots of ginseng were treated with methyl jasmonate (MJ) up to 150m and cultured for 40days. Up to 100m MJ inhibited the root growth but increase ginsenoside accumulation. In a two-stage bioreactor culture, total ginsenosides, after elicitation with 100 m MJ peaked after 10days at 48mgg^–1 dry wt and then dropped sharply. Of the two groups of ginsenosides (Rb and Rg), higher amounts of Rb accumulated in the adventitious roots.Revisions requested; 2 July 2004; Revisions received 30 June 2004; 3 September 2004     

Effect of sugar concentration on ginsenoside biosynthesis in hairy root cultures of Panax quinquefolium cultivated in shake flasks and nutrient sprinkle bioreactor

The study assessed the influence of sugar concentration (10, 20, 30, 50, 70, 100, 120 g l^?1) on growth and ginsenoside biosynthesis in Panax quinquefolium hairy roots cultivated in shake flasks and a nutrient sprinkle bioreactor. The highest growth rate was achieved in medium containing 3–5 % sucrose. More than 70 g l^?1 or less than 20 g l^?1 sugar content in the medium induces significant inhibition of root growth when cultivated in shake flasks. The saponin content was determined using HPLC. The maximum yield (above 9 mg g^?1 d.w.) of the sum of six examined ginsenosides (Rb₁, Rb₂, Rc, Rd, Re and Rg₁) in hairy roots cultivated in shake flasks was obtained with 30 g l^?1 sucrose in the medium. The sucrose concentration in the medium was found to correlate with saponin content in bioreactor-cultured specimens. A higher level of protopanaxadiol derivatives was found for lower (20 and 30 g l^?1) sucrose concentrations; higher sucrose concentrations (50 and 70 g l^?1) in the medium stimulated a higher level of Rg group saponins.     

Effect of auxins on <Emphasis Type="Italic">Karwinskia humboldtiana</Emphasis> root cultures

Effects of auxins (IAA, IBA and NAA) on K. humboldtiana root culture cultivated in 16-h photoperiod or in dark have been observed. Light affected positively the production of biomass when cultivated on medium supplemented with NAA in 10 and 25 mol ^–1 concentrations. In the presence of IAA and IBA these values were significantly lower. The growth dynamics of root cultures depended on the auxin used. The best adventitious roots elongation and lateral roots induction on media supplemented with IBA has been ascertained. Morphological and anatomical differences in dependence on auxin used were observed. NAA supported the formation of huge callus-like mass besides mostly very short roots, especially under the light. Similarly IAA induced short roots, and IBA seems to be the most effective substance for the root elongation in this model system. NAA induced roots with larger diameter under the light compared with the other two auxins used. The reason is in the different anatomical structure of roots which was characterized by higher number of cell layers and large intercellulars in the cortex. The shape of cortical cells in the presence of IBA depended on the light conditions. Isodiametric cortical cells were present in roots cultivated in 16-h photoperiod, irregularly-shaped cells in the dark. The effect of light conditions was the smallest in the case of roots grown on IAA enriched media.     

Inoculum size and auxin concentration influence the growth of adventitious roots and accumulation of ginsenosides in suspension cultures of ginseng ( Panax ginseng C.A. Meyer)

The effect of the root-inoculum size and axuin concentration on growth of adventitious roots and accumulation of ginsenosides were studied during suspension cultures of ginseng (Panax ginseng C.A. Meyer). Of the various concentrations of indole-3-butyric acid (IBA) and γ-naphthaleneacetic acid (NAA) used as supplementary growth regulators along with Murashige and Skoog medium, 25 μM IBA was found suitable for lateral root induction and growth, as well as accumulation of ginsenosides. Inoculum size of 5 g L⁻¹ was found suitable for optimal biomass (10.5 g L⁻¹ dry biomass) and ginsenosides (5.4 mg g⁻¹ DW) accumulation. Of the various length of root inocula tested (chopped to 1–3, 4–6, 7–10 mm and un-chopped), root inocula of 7–10 mm was found suitable for biomass and ginsenoside accumulation.     

Podophyllotoxin production via cell and adventitious root cultures of <Emphasis Type="Italic">Podophyllum peltatum</Emphasis>

Podophyllum peltatum is an important medicinal plant that produces podophyllotoxin (PTOX) with anti-cancer properties. We established the embryogenic cell and adventitious root culture systems in P. peltatum and analyzed PTOX production. For the growth of embryogenic cell clumps in shake flask culture, the most efficient concentration of 2,4-dichloroacetic acid (2,4-D) was 6.78 μM, and the growth of embryogenic cell clumps was 15.9-fold increased in Murashige and Skoog MS liquid medium with 6.78 μM 2,4-D after 3 wk of culture. To induce adventitious roots, half-strength MS medium showed the best results for adventitious root induction compared to full strength MS medium and MS medium lacking NH₄NO₃. Optimal indole-3-butyric acid concentration for adventitious root formation was 14.78 μM. In liquid medium, the frequency of adventitious root formation from root segments was 87.7% and the number of laterally formed adventitious roots was 22.3 per segment. PTOX production in embryogenic cells and adventitious roots was confirmed by liquid chromatography and electrospray ionization–tandem mass spectrometry analysis. High-performance liquid chromatography analysis revealed that adventitious roots contained higher PTOX than embryogenic cell clumps. Elicitor treatment (20 μM methyl jasmonate) strongly enhanced the production of PTOX in both embryogenic cell clumps and adventitious roots. This observation suggests that both embryogenic cell and adventitious root culture can be adopted to produce PTOX.     

Growth,secondary metabolite production and antioxidant enzyme response of <Emphasis Type="Italic">Morinda citrifolia</Emphasis> adventitious root as affected by auxin and cytokinin

Morinda citrifolia adventitious roots were cultured in shake flasks using Murashige and Skoog medium with different types and concentrations of auxin and cytokinin. Root (fresh weight and dry weight) accumulation was enhanced at 5 mg l⁻¹ indole butyric acid (IBA) and at 7 and 9 mg l⁻¹ naphthalene acetic acid (NAA). On the other hand, 9 mg l⁻¹ NAA decreased the anthraquinone, phenolic and flavonoid contents more severely than 9 mg l⁻¹ IBA. When adventitious roots were treated with kinetin (0.1, 0.3 and 0.5 mg l⁻¹) and thidiazuron (TDZ; 0.1, 0.3 and 0.5 mg l⁻¹) in combination with 5 mg l⁻¹ IBA, fresh weight and dry weight decreased but secondary metabolite content increased. The secondary metabolite content (including 1,1-diphenyl-2-picrylhydrazyl activity) increased more in TDZ-treated than in kinetin-treated roots. Antioxidative enzymes such as catalase (CAT) and guaiacol peroxidase (G-POD), which play important roles in plant defense, also increased. A strong decrease in ascorbate peroxidase activity resulted in a high accumulation of hydrogen peroxide. This indicates that adventitious roots can grow under stress conditions with induced CAT and G-POD activities and higher accumulations of secondary metabolites. These results suggest that 5 mg l⁻¹ IBA supplementation is useful for growth and secondary metabolite production in adventitious roots of M. citrifolia.