Transient studies of nutrient uptake, growth, and indole alkaloid accumulation in heterotrophic cultures of hairy roots of Catharanthus roseus

The kinetics of growth, the uptake of macronutrients, and the accumulation of indole alkaloids were investigated in long-term, heterotrophically cultured transgenic ("hairy") roots of Catharanthus roseus.Tabersonine, ajmalicine, and serpentine were monitored over a 70-day period. The doubling time [dry-weight (DW) basis] of C. roseus hairy roots in B5/2 nutrients supplemented with 3% sucrose was 3.6 days. NH(4) (+), NO(3),(-) and P(i) were depleted sequentially from culture medium by hairy roots, while sugars remained undepleted. The growth-limiting nutrient was inorganic nitrogen, NH(4) (+) and NO(3) (-), with exponential-phase overall biomass yields of 34.1 and 5.0 g DW/g nutrient, respectively. Extracellular pH decreased to 4.8 in early exponential phase of culture growth from the initially adjusted value of 5.7, increased subsequently to a maximum of 7.7 in late exponential phase of growth coincident with the maximum of fresh weight (FW)/DW ratio, before decreasing to 5.5-5.0. The organic acids, pyruvate, formate, lactate, and succinate were excreted by hairy roots starting in late phase of exponential growth, possibly resulting in the late-culture pH decrease. Tabersonine accumulation was distinctly growth associated with maximum specific and total yields of 1.15 mg/g DW and 5.6 mg/L, respectively, in late-exponential phase of growth. Serpentine accumulation was non growth associated with increasing specific and total levels in stationary growth phase: 1.3 mg/g DW and 10.5 mg/L, respectively. The accumulation of ajmalicine also appeared growth associated. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 527-534, 1997.     

莴笋对不同形态氮素的反应

探讨了不同形态氮素对莴笋生长发育的影响及其营养特性。结果表明,莴笋幼苗根系对NH4^+ -N的亲和力稍大于NO3^- -N的亲和力;分别供给NO3^- -N+NO3^- -N及NH4^+ -N,莴笋的生物学产量和吸N量均依次递减（分别为100：56.9：12.4,100：48.9：8.6）,因此在水培条件下,NO3^- -N是最适合莴笋生长发育的氮源,NH4^+ -N与NO3^- -N各占50％时对莴笋的生长发育已有一定的抑制作用,仅以NH4^+ -N作氮源则莴笋很难生长;NH4^+ -N与NO3^- -N各占50％时,莴笋倾向于吸收较多的NH4^+ -N,而且在培养不同阶段NH4^+/NO3^-吸收比例均大于1,莴笋表现出喜铵性,但NH4^+ -N并非莴笋很适合的氮源;营养液中NO3^- -N不足,主要影响莴笋茎叶的生长,而NH4^+ -N所占比例达50％时,莴笋根系生长受到抑制,且有明显的受害症状;以NO3^- -N作氮源预培养两周,以含微量NO3^- -N的自来水为水源,再单独以NH4^+ -N为氮源,对莴笋生长有极大的促进作用,同时还大幅度降低了体内硝酸盐的含量。尿素作氮源莴笋未出现受害症状,但莴笋的生长发育状况明显劣于其它氮源。     

褐脉少花龙葵毛状根的诱导、培养及其澳洲茄胺的产生

利用发根农杆茵的遗传转化和液体培养技术,研究了褐脉少花龙葵(solanum nigrum L.Var.Dauciforum)毛状根的诱导和离体培养及其澳洲茄胺的产生以及液体培养过程中培养基中N源和钙的消耗变化.结果表明.发根农杆菌ATCC15834感染褐脉少花龙葵叶片外植体5 d后产生毛状根.感染25d后,约90%的叶片外植体产生毛状根.毛状根能在无外源生长调节剂的MS固体和体培养基上自主生长.PCR扩增结果显示发根农杆菌Ri质粒的rilB和rolC基因已在少花龙葵毛状根基因组中整合并得到表达.所产生的毛状根能产生药用次生物质澳洲茄胺,其含量约为非转化植株根的1.3倍,达到582.05μg/g干重.少花龙葵毛状根液体培养0-5 d内处于生长迟滞期、5-15 d为快速生长期、15d后进入生长平台期.培养基的硝态氮和铵态氮在毛状根液体培养过程中被逐渐吸收和消耗,至培养15 d时铵态氮被消耗殆尽.而硝态氮仍剩余44.7%;培养基中钙的浓度在培养过程中虽逐渐降低,但在培养25d时仍未被完全消耗,其浓度约为起始浓度的43.5%.该结果为今后设计合适的培养基来规模培养褐脉少花龙葵毛状根生产药用次生物质澳洲茄胺提供了可能性.     

The effect of nitrate and ammonium concentrations on growth and alkaloid accumulation of Atropa belladonna hairy roots

The growth, the alkaloid production, as well as the scopolamine/hyoscyamine ratio of two clones of belladonna hairy roots were studied. The effects of nitrate and ammonium concentrations on these cultures were investigated. A rise in ammonium concentration caused the decline of the hairy roots, while nitrate had a marked effect on the alkaloid content. The alkaloid production obtained with 15.8 mM of NO3- and 20.5 mM of NH4+ was 1.2-1.4 times higher than that obtained when the roots were grown in the standard Murashige and Skoog medium (MS medium, 39.5 mM of NO3- and 20.5 mM of NH4+). The nitrate and ammonium concentrations in the culture medium also had a strong influence on the scopolamine/hyoscyamine ratio. When nitrate or ammonium concentrations were raised, that ratio also was increased 2-3-fold. The hairy root clones originating from transformations with two distinct strains of Agrobacterium had similar responses.     

Ammonium and nitrate uptake by soybean during recovery from nitrogen deprivation

Soybean [Glycine max (L.) Merrill] plants that had been subjected to 15 d of nitrogen deprivation were resupplied for 10 d with 1.0 mol m-3 nitrogen provided as NO3-, NH4+, or NH4(+) + NO3- in flowing hydroponic culture. Plants in a fourth hydroponic system received 1.0 mol m-3 NO3- during both stress and resupply periods. Concentrations of soluble carbohydrates and organic acids in roots increased 210 and 370%, respectively, during stress. For the first day of resupply, however, specific uptake rates of nitrogen, determined by ion chromatography as depletion from solution, were lower for stressed than for non-stressed plants by 43% for NO3- resupply, by 32% for NH4(+) + NO3- resupply, and 86% for NH4+ resupply. When specific uptake of nitrogen for stressed plants recovered to rates for non-stressed plants at 6 to 8 d after nitrogen resupply, carbohydrates and organic acids in their roots had declined to concentrations lower than those of non-stressed plants. Recovery of nitrogen uptake capacity of roots thus does not appear to be regulated simply by the content of soluble carbon compounds within roots. Solution concentrations of NH4+ and NO3- were monitored at 62.5 min intervals during the first 3 d of resupply. Intermittent 'hourly' intervals of net influx and net efflux occurred. Rates of uptake during influx intervals were greater for the NH4(+)-resupplied than for the NO3(-)-resupplied plants. For NH4(+)-resupplied plants, however, the hourly intervals of efflux were more numerous than for NO3(-)-resupplied plants. It thus is possible that, instead of repressing NH4+ influx, increased accumulation of amino acids and NH4+ in NH4(+)-resupplied plants inhibited net uptake by stimulation of efflux on NH4+ absorbed in excess of availability of carbon skeletons for assimilation. Entry of NH4+ into root cytoplasm appeared to be less restricted than translocation of amino acids from the cytoplasm into the xylem.     

培养基磷缺乏对黄瓜毛状根生长、抗氧化酶活性及氮源利用的影响

采用液体培养的方法研究了培养基磷缺乏对黄瓜毛状根生长及其抗氧化酶活性及培养基中氮源和钙利用的影响.结果表明,黄瓜毛状根在完全缺磷的培养基中几乎不能生长;而培养基无机磷缺乏会抑制黄瓜毛状根的生长,且浓度越低,其抑制作用越明显,毛状根变得越纤细而长,侧根数减少且短小.与全磷培养相比,磷缺乏培养基培养的黄瓜毛状根可溶性蛋白含量明显偏低,但其SOD和POD活性则明显升高.与完全缺磷(对照)相比,在培养过程中不同无机磷浓度培养的黄瓜毛状根的SOD和POD活性均比对照低.当黄瓜毛状根在不同磷缺乏浓度的液体培养基中培养时,随着培养时间的延长,培养基的电导率逐步下降,并与培养基起始无机磷浓度成正比;其培养基的铵态氮和硝态氮不断被吸收和利用,培养至15d时,培养基中的铵态氮已绝大部分被消耗完毕,但直至培养30d时培养基的硝态氮仍未被消耗完毕.培养基中无机磷缺乏会降低黄瓜毛状根对培养基硝态氮的吸收和消耗以及抑制黄瓜毛状根对钙的吸收.而适当提高培养基的无机磷浓度可促进黄瓜毛状根对培养基中钙的吸收和消耗.     

Artemisinin production in Artemisia annua hairy root cultures with improved growth by altering the nitrogen source in the medium

Murashige & Skoog medium was modified for enhancing artemisinin production in Artemisia annua hairy root cultures by altering the ratio of NO ₃ ^– /NH ₄ ⁺ and the total amount of initial nitrogen. Increasing ammonium to 60 mM decreased both growth and artemisinin accumulation in hairy root cultures. With NO ₃ ^– /NH ₄ ⁺ at 5:1 (w/w), the optimum concentration of total initial nitrogen for artemisinin production was 20 mM. After 24 days of cultivation with 16.7 mM nitrate and 3.3 mM ammonium, the maximum artemisinin production of hairy roots was about 14 mg l^–1, a 57% increase over that in the standard MS medium.     

An interchangeable system of hairy root and cell suspension cultures ofCatharanthus roseus for indole alkaloid production

Hairy roots ofCatharanthus roseus obtained by co-cultivation of hypocotyl segments withAgrobacterium rhizogenes, and cultured in SH (Schenk and Hildebrandt) basal medium, formed two types of calli when subcultured in SH medium with 1 mg/1 -naphthaleneacetic acid and 0.1 mg/l kinetin. One of them, a compact callus, when re-subcultured in SH basal medium gave rise to hairy roots again. A rhizogenic cell suspension culture was established from this type of callus. When cultured in SH medium with growth regulators, the rhizogenic callus produced catharanthine at a level of 41% of the level in the initial hairy roots. Upon transfer to SH basal medium, regenerated hairy roots produced this alkaloid at the original level of 1.5 mg/g dry wt. Using this cell/hairy root interchange system a new management system for hairy root culture in bioreactors has been devised and examined involving production of biomass in the form of a cell suspension in medium supplemented with growth regulators, and catharanthine production by hairy roots regenerated from these cells in medium without growth regulators.Abbreviations NAA -naphthaleneacetic acid - SH Schenk and Hildebrandt - SHNK SH medium + 1 mg 1^–1 NAA + 0.1 mg 1^–1 kinetin     

培养条件对新疆紫草毛状根生长及紫草素含量的影响

以发根农杆菌诱导的新疆紫草毛状根为试验材料,采用二阶段液体培养法,首次建立了新疆紫草毛状根培养技术体系。结果显示:采用SH无铵培养基、pH 5.8时有利于毛状根的生长。培养12d时毛状根的增殖倍数达最高,平均10.26倍;毛状根生产的继代周期为25～30d;4种树脂吸附的紫草素及其衍生物含量均较对照(不添加树脂)高,以NKA-9所吸附的紫草素及其衍生物含量最高,为2.38%,较对照提高0.97倍。培养10d时添加NKA-9树脂,紫草素及其衍生物含量平均为3.64%,是对照的3.08倍。研究表明,生长阶段采用液体培养可以使新疆紫草毛状根快速增殖,生产阶段添加大孔吸附树脂能够提高紫草素及其衍生物含量。     

Hairy roots of Dracocephalum moldavica: rosmarinic acid content and antioxidant potential

Hairy roots of Dracocephalum moldavica L. were induced using Agrobacterium rhizogenes strain A4. Transformed roots were obtained from shoot explants with low transformation frequency of up to 3 %. The effects of different liquid media: Murashige and Skoog (MS), Gamborg et al. (B5) and Woody Plant (WP) with full- and half-strength (½MS, ½B5, ½WP), on biomass accumulation and rosmarinic acid (RA) content were investigated. The hairy roots were cultured in photoperiod (16 h light/8 h dark) and darkness. Biomass of D. moldavica hairy roots was the highest (7.23 g flask^?1 of fresh weight and 0.89 g flask^?1 of dry weight) in the cultures grown in WP medium under periodic light. Ultra performance liquid chromatography analysis revealed the highest RA content (78 mg g^?1 dry wt) in roots cultured in ½B5 medium under photoperiod conditions. It was about tenfold higher compared to roots of field-grown mother plants. Antioxidant activities and total phenolic contents of methanolic extracts of D. moldavica hairy roots cultured in ½B5 and WP media under photoperiod and darkness and roots of field grown plants were compared. All extracts were investigated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging and phosphomolybdenum reduction assays. Total phenolic contents were estimated by the Folin–Ciocalteu method. The methanolic extract of D. moldavica hairy roots grown in ½B5 medium under photoperiod possessed the strongest effects on reducing Mo and DPPH radical scavenging. The activities were significantly higher (p ≤ 0.05) than those of methanolic extract of roots of intact plants grown in the field. The most active methanolic extract of hairy roots was characterized by the highest level of rosmarinic acid and total content of phenolic compounds.     

Effect of nitrogen source on biomass and bioactive compound production in submerged cultures of Eleutherococcus koreanum Nakai adventitious roots

Ammonium to nitrate ratios of 0:30, 5:25, 10:20, 15:15, 20:10, 25:5, and 30:0 mM were tested to determine the optimal NH(4)(+) :NO(3)(-) ratio for improving biomass and bioactive compound production in Eleutherococcus koreanum Nakai adventitious roots using 3-L bulb-type bubble bioreactors. A high ammonium nitrogen ratio had a negative effect on root growth, and the highest fresh and dry weights were obtained when NH(4)(+):NO(3)(-) ratios were 5:25 and 10:20 (mM) after 5 weeks of culture. Although the total production of eleutherosides B and E was slightly higher at the 10:20 ratio than at the 5:25 ratio (NH(4)(+):NO(3)(-)), we proposed that the optimal NH(4)(+):NO(3)(-) ratio was 5:25 mM. This ratio achieved both the highest total production of five target bioactive compounds (eleutherosides B and E, chlorogenic acid, total phenolics, and flavonoids) and the highest root biomass. Furthermore, increasing NH(4)(+):NO(3)(-) ratios to 10:20 decreased pH in the medium, interrupted the absorption of essential minerals from the culture medium, and resulted in low biomass and increased relative oxidative stress levels, which were evaluated by determining 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. Therefore, nitrate rather than ammonium nitrogen was more essential not for only biomass production but also for bioactive compound production in E. koreanum adventitious root cultures. The optimal nitrogen source ratio produced 5.63 g L(-1) of biomass and 24.41 mg of the five total bioactive compounds per gram of biomass (dry weight basis). The development of such in vitro culture technology will benefit the pilot-scale production of E. koreanum-based bioactive compounds for commercialization.     

Partial substitution of NO(3)(-) by NH(4)(+) fertilization increases ammonium assimilating enzyme activities and reduces the deleterious effects of salinity on the growth of barley

Productivity of cereal crops is restricted in saline soils but may be improved by nitrogen nutrition. In this study, the effect of ionic nitrogen form on growth, mineral content, protein content and ammonium assimilation enzyme activities of barley (Hordeum vulgare cv. Alexis L.) irrigated with saline water, was determined. Leaf and tiller number as well as plant fresh and dry weights declined under salinity (120 mM NaCl). In non-saline conditions, growth parameters were increased by application of NH(4)(+)/NO(3)(-) (25:75) compared to NO(3)(-) alone. Under saline conditions, application of NH(4)(+)/NO(3)(-) led to a reduction of the detrimental effects of salt on growth. Differences in growth between the two nitrogen regimes were not due to differences in photosynthesis. The NH(4)(+)/NO(3)(-) regime led to an increase in total N in control and saline treatments, but did not cause a large decrease in plant Na(+) content under salinity. Activities of GS (EC 6.3.1.2), GOGAT (EC 1.4.1.14), PEPC (EC 4.1.1.31) and AAT (EC 2.6.1.1) increased with salinity in roots, whereas there was decreased activity of the alternative ammonium assimilation enzyme GDH (EC 1.4.1.2). The most striking effect of nitrogen regime was observed on GDH whose salinity-induced decrease in activity was reduced from 34% with NO(3)(-) alone to only 14% with the mixed regime. The results suggest that the detrimental effects of salinity can be reduced by partial substitution of NO(3)(-) with NH(4)(+) and that this is due to the lower energy cost of N assimilation with NH(4)(+) as opposed to NO(3)(-) nutrition.     

Effect of nitrogen form and root-zone pH on growth and nitrogen uptake of tea (Camellia sinensis) plants

BACKGROUND AND AIMS: Tea (Camellia sinensis) is considered to be acid tolerant and prefers ammonium nutrition, but the interaction between root zone acidity and N form is not properly understood. The present study was performed to characterize their interaction with respect to growth and mineral nutrition. METHODS: Tea plants were hydroponically cultured with NH4+, NO3- and NH(4+) + NO3-, at pH 4.0, 5.0 and 6.0, which were maintained by pH stat systems. KEY RESULTS: Plants supplied with NO3- showed yellowish leaves resembling nitrogen deficiency and grew much slower than those receiving NH4+ or NH(4+) + NO3- irrespective of root-zone pH. Absorption of NH4+ was 2- to 3.4-fold faster than NO3- when supplied separately, and 6- to 16-fold faster when supplied simultaneously. Nitrate-grown plants had significantly reduced glutamine synthetase activity, and lower concentrations of total N, free amino acids and glucose in the roots, but higher concentrations of cations and carboxylates (mainly oxalate) than those grown with NH4+ or NH(4+) + NO3-. Biomass production was largest at pH 5.0 regardless of N form, and was drastically reduced by a combination of high root-zone pH and NO3-. Low root-zone pH reduced root growth only in NO(3-)-fed plants. Absorption of N followed a similar pattern as root-zone pH changed, showing highest uptake rates at pH 5.0. The concentrations of total N, free amino acids, sugars and the activity of GS were generally not influenced by pH, whereas the concentrations of cations and carboxylates were generally increased with increasing root-zone pH. CONCLUSIONS: Tea plants are well-adapted to NH(4+)-rich environments by exhibiting a high capacity for NH4+ assimilation in their roots, reflected in strongly increased key enzyme activities and improved carbohydrate status. The poor plant growth with NO3- was largely associated with inefficient absorption of this N source. Decreased growth caused by inappropriate external pH corresponded well with the declining absorption of nitrogen.     

Root-zone acidity and nitrogen source affects Typha latifolia L. growth and uptake kinetics of ammonium and nitrate

The NH(4)(+) and NO(3)(-) uptake kinetics by Typha latifolia L. were studied after prolonged hydroponics growth at constant pH 3.5, 5.0, 6.5 or 7.0 and with NH(4)(+) or NO(3)(-) as the sole N-source. In addition, the effects of pH and N source on H(+) extrusion and adenine nucleotide content were examined. Typha latifolia was able to grow with both N sources at near neutral pH levels, but the plants had higher relative growth rates, higher tissue concentrations of the major nutrients, higher contents of adenine nucleotides, and higher affinity for uptake of inorganic nitrogen when grown on NH(4)(+). Growth almost completely stopped at pH 3.5, irrespective of N source, probably as a consequence of pH effects on plasma membrane integrity and H(+) influx into the root cells. Tissue concentrations of the major nutrients and adenine nucleotides were severely reduced at low pH, and the uptake capacity for inorganic nitrogen was low, and more so for NO(3)(-)-fed than for NH(4)(+)-fed plants. The maximum uptake rate, V(max), was highest for NH(4)(+) at pH 6.5 (30.9 micro mol h(-1) g(-1) root dry weight) and for NO(3)(-) at pH 5.0 (31.7 micro mol h(-1) g(-1) root dry weight), and less than 10% of these values at pH 3.5. The affinity for uptake as estimated by the half saturation constant, K((1/2)), was lowest at low pH for NH(4)(+) and at high pH for NO(3)(-). The changes in V(max) and K((1/2)) were thus consistent with the theory of increasing competition between cations and H(+) at low pH and between anions and OH(-) at high pH. C(min) was independent of pH, but slightly higher for NO(3)(-) than for NH(4)(+) (C(min)(NH(4)(+)) approximately 0.8 mmol m(-3); C(min)(NO(3)(-)) approximately 2.8 mmol m(-3)). The growth inhibition at low pH was probably due to a reduced nutrient uptake and a consequential limitation of growth by nutrient stress. Typha latifolia seems to be well adapted to growth in wetland soils where NH(4)(+) is the prevailing nitrogen compound, but very low pH levels around the roots are very stressful for the plant. The common occurrence of T. latifolia in very acidic areas is probably only possible because of the plant's ability to modify pH-conditions in the rhizosphere.     

Effect of ammonium and nitrate on growth and appearance of nitrate reductase and nitrite reductase in dark- and light-grown mustard seedlings

Nitrate-induced and phytochrome-modulated appearance of nitrate reductase (NR; EC 1.6.6.1) and nitrite reductase (NIR; EC 1.7.7.1) in the cotyledons of the mustard (Sinapis alba L.) seedling is strongly affected by externally supplied ammonium (NH ₄ ⁺ ). In short-term experiments between 60 and 78 h after sowing it was found that in darkness NH ₄ ⁺ —simultaneously given with NO ₃ ^- —strongly inhibits appearance of nitrate-inducible NR and NIR whereas in continuous far-red light—which operates exclusively via phytochrome without significant chlorophyll formation —NH ₄ ⁺ (simultaneously given with NO ₃ ^- ) strongly stimulates appearance of NR. The NIR levels are not affected. This indicates that NR and NIR levels are regulated differently. In the absence of external NO ₃ ^- appearance of NR is induced by NH₄ in darkness as well as in continuous far-red light whereas NIR levels are not affected. On the other hand, in the absence of external NO ₃ ^- , exogenous NH ₄ ⁺ strongly inhibits growth of the mustard seedling in darkness as well as in continuous far-red light. This effect can be abolished by simultaneously supplying NO ₃ ^- . The adverse effect of NH ₄ ⁺ on growth (NH ₄ ⁺ -toxicity) cannot be attributed to pH-changes in the medium since it was shown that neither the growth responses nor the changes of the enzyme levels are related to pH changes in the medium. Non-specific osmotic effects are not involved either.Abbreviations c continuous - D darkness - FR far-red light - NIR nitrite reductase (EC 1.7.7.1) - NR nitrate reductase (EC 1.6.6.1)     

Establishment of Hairy Root Cultures of Astragalus membranaceus and the Extrinsic Factors Affecting Their Growt

Hairy root cultures were established from the leaf explant of Astragalus membranaceus (Fisch.) Bge. infected with Agrobacterium rhizogenes and factors that affect their growth were investigated. It was found that the hairy roots grew best in a phytohonnone-free MS medium at pH 6, in which the most optimal concentration of sucrose was 3 % and NH4NO3 was omitted because of its inhibitory effect to the hairy roots. Light was also inhibitory to them but the effect of low temperature was not apparent. Moreover, there were more crude saponin and soluble polysuccharide contents in the hairy roots than in the natural dry roots. Finally, a discussion on the application and the potenrial of the hairy root technique in the production of the plant roots as an effection Chinese traditional medicine was also involved.     

Hairy root culture of <Emphasis Type="Italic">Plumbago indica</Emphasis> as a potential source for plumbagin

Hairy roots of Plumbago indica were established at high frequency (90 %) by infecting leaf explants with Agrobacterium rhizogenes strain ATCC 15834. The axenic root cultures were established under darkness in hormone-free liquid Murashige and Skoog medium containing 3 % sucrose. The highest plumbagin content was found to accumulate in roots at their exponential phase of growth. A low pH (4.6) and a low concentration of sucrose (1 %) were beneficial for root growth in darkness, while pH 5.6 and 3 % sucrose under continuous irradiance enhanced plumbagin accumulation in roots up to 7.8 mg g⁻¹(d.m.). Direct shoot regeneration from hairy root culture was also achieved under continuous irradiance, thus indicated an easy way of obtaining transformed P. indica plants.     

野葛毛状根离体培养与异黄酮生产

用发根农杆菌（Agrobacterium rhizogenes）R1601菌株感染野葛（Pueraria lobata(Willd.)Ohwi）叶片外植体后获得的毛状根,经连续4次继代培养,鲜重增加到最初的6.2倍。PH为5.5的1/2SH液体培养基较适合野葛毛状根的生长。与自然根相比,毛状根在无激素的1/2SH液体培养基中离体培养20d后,鲜重增加22.6倍,释放入培养液中的异黄酮量增加10.6倍。毛状根生产异黄酮的量略高于自然根,且根中含量明显高于愈伤组织及茎中异黄酮的含量。培养液体积影响毛状根的离体生长及异黄酮的释放。