Variation in pigment production in Lithospermum erythrorhizon callus cultures

Different strains of callus cultures of Lithospermum erythrorhizon showed wide variations in the production of shikonin derivatives. From these cultures, two high pigment-producing strains, whose content of shikonin derivatives are stable and similar to that of intact plant root, have been established by repeated selection.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon

Differences in the production of shikonin derivatives by callus and suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. were examined. When Linsmaier and Skoog medium was used in suspension cultures, cell growth was not accompanied by the production of shikonin compounds. Shikonin derivatives were produced, however, when this medium was used in callus cultures. Differences in shikonin production were examined in terms of the nutrient supply, the effect of the agar itself, and the oxygen supply. Shikonin derivatives could be produced without agar by keeping the cells exposed to air while providing an adequate supply of nutrients. In callus cultures, the production of shikonin compounds was reduced remarkedly when the oxygen concentration in the atmosphere was lowered, evidence that shikonin production during L. erythrorhizon cell growth on Linsmaier and Skoog agar medium is enhanced by an abundant supply of oxygen.     

Pigment formation in callus cultures of Lithospermum erythrorhizon

Undifferentiated callus tissues of Lithospermum erythrorhizon are capable of synthesizing shikonin derivatives, which are normally formed in the cork cells of the roots. Their biosynthesis in cultured cells is controlled by auxin and light. The pigment content increased linearly with time after a lag phase when callus tissues were grown on culture medium containing IAA in the dark, whereas it markedly decreased when 2,4-D was substituted for IAA or when cultures were irradiated with blue light.     

Biosynthesis of shikonin in callus cultures of Lithospermum erythrorhizon

Administration of various supposed precursors to the callus cultures of Lithospermum erythrorhizon grown on the Linsmaier—Skoog medium supplemented with IAA and kinetin established that the constituent shikonin is formed via shikimic acid, p-hydroxybenzoic acid, m-geranyl-p-hydroxybenzoic acid and geranylhydroquinone. In a strain of callus culture lacking the capacity to synthesize shikonin and in callus cultures which have had this capacity but lost it due to cultivation on a medium supplemented with 2,4-D, substances up to m-geranyl-p-hydroxybenzoic acid in the biosynthetic sequence have been detected. Although illumination with white light also arrested shikonin production, traces of pigment were still formed presumably because light did not reach the innermost part of the callus cultures.     

In Vitro Long-Term Storage of Date Palm

A reliable method for the long-term conservation of date palm tissue cultures is described. In vitro shoot bud and callus culture were successfully stored for 12 months at 5°C in the dark. At this conditions high percent of cultures remained viable without serious signs of senescence. However, the growth rate decreased as storage period increased. The role of sorbitol as osmotic agent in storage was examined. Health shoot bud cultures were obtained after 6 months of storage on medium containing 40 g dm^–3 sorbitol. This period extended for 9 months in case of callus cultures.     

Stable transformation of Lithospermum erythrorhizon by Agrobacterium rhizogenes and shikonin production of the transformants

Seedling hypocotyls of Lithospermum erythrorhizon were infected with Agrobacterium rhizogenes (strain 15834) harboring a binary vector with an intron-bearing the β-glucuronidase (GUS) gene driven by cauliflower mosaic virus (CaMV) 35S promoter as well as the hygromycin phosphotransferase (HPT) gene as the selection marker. About 20% of the hairy roots isolated were hygromycin resistant and had co-integrated GUS and HPT genes in their Lithospermum genomic DNA. Because GUS activity was detected in almost all the hygromycin-resistant root tissues, the CaMV 35S promoter seems to be ubiquitously active in L. erythrorhizon hairy roots. In pigment production medium M9, the hairy root cultures had shikonin productivity similar to that of cell suspension cultures of Lithospermum. They also showed light-dependent inhibition of shikonin biosynthesis similar to that of Lithospermum cell cultures. These findings suggest that this hairy root system transformable with A. rhizogenes is a suitable model system for molecular characterization of shikonin biosynthesis via reverse genetics. Received: 2 March 1998 / Revision received: 25 May 1998 / Accepted: 8 July 1998     

Increased shikonin production inLithospermum erythrorhizon suspension cultures within situ extraction and fungal cell treatment (elicitor)

Summary The effect ofin situ extraction and elicitor treatment on shikonin production was studied with the suspension cultures ofLithospermum erythrorhizon. Shikonin concentration of 60 mg/L was achieved by the use of both techniques which was 24 times higher than that of control culture, and 65 times higher in terms of shikonin productivity. The host-pathogen effect of elicitor treatment andin situ extraction for product removal were effective for shikonin production.     

柴胡幼苗越冬抗寒性及其相关生理指标筛选

以北柴胡、地产柴胡、三岛柴胡的根为试验材料,以早春植株成活率为越冬抗寒性指标,测定越冬期不同柴胡品种根系生理生化特征,进行柴胡抗寒性综合评价,并对主要抗寒生理指标进行通径分析和相关性分析,以探讨冬季自然低温条件下柴胡生理特性与抗寒性的关系,筛选适合柴胡越冬抗寒性鉴定的生理指标。结果表明:(1)北柴胡、地产柴胡、三岛柴胡返青期的返青率依次为80%、50%、10%,且各品种间差异显著。(2)3个品种柴胡越冬期根系可溶性糖含量、游离脯氨酸含量、可溶性蛋白含量、根系活力与返青率大小顺序一致,其中北柴胡根系游离脯氨酸平均含量分别为地产柴胡和三岛柴胡的2.44倍和4.49倍,且可溶性蛋白含量显著高于地产柴胡、三岛柴胡,但3个品种柴胡的相对电导率、丙二醛含量水平与返青率大小顺序相反。(3)自然越冬过程中,各柴胡品种的根系活力均呈下降趋势,且以三岛柴胡根系活力下降幅度最大,达80%,而北柴胡根系活力下降幅度最小为52.04%。(4)越冬期3个柴胡品种的越冬抗寒性综合排序为:北柴胡地产柴胡三岛柴胡;柴胡根中脯氨酸含量、可溶性蛋白含量与综合评价值呈极显著正相关关系,相对电导率与综合评价值呈显著负相关关系;生理指标决策系数的大小顺序依次为脯氨酸含量可溶性蛋白含量电导率。研究表明,越冬期柴胡根系游离脯氨酸含量是影响其抗寒性的主要影响因素,其含量可作为评价柴胡抗寒性的指标。     

Callus formation from protoplasts of cultured Lithospermum erythrorhizon cells

Protoplasts isolated from cell cultures of Lithospermum erythrorhizon divided repeatedly and formed callus colonies. Factors that affect protoplast division are the use of glucose as osmoticum, a new plating method with twin layers of agar-liquid medium, and the culture of protoplasts under the osmolarity lower than that in the isolation solution. When the sucrose in the protoplast-culture medium was replaced with glucose, and coconut milk was added to the medium, the frequency of colony formation markedly increased. The culture period required for colony formation also was shortened.     

Effect of growth hormone modifications on shikonin production fromLithospermum erythrorhizon cell cultures within situ extraction

Summary The effect of growth hormone modifications on shikonin production was studied with the cell cultures ofLithospermum erythrorhizon. The cells grown in SH–H or SHA medium were effective for shikonin production in M–9 medium and maximum shikonin concentrations reached 43 and 63 mg/L, respectively, within situ extraction. In the case of the cells grown in SHA medium, induction time required for shikonin production was very short and the maximum shikonin concentration was obtained within 6 days.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon

An excellent new medium was developed for the production of shikonin derivatives by suspension cultures of Lithospermum erythrorhizon. We investigated the effects of all the components of White's medium on the production of these derivatives. Nitrate, phosphate, copper, sulfate and sucrose had especially marked effects. With the new, M-9, medium produced from these studies the yield of shikonin derivatives was 1400 mg/l and the yield for dried cells was about 12%, whereas it was 120 mg/l, or about 2% with White's medium.     

Two phenolic acids from Lithospermum erythrorhizon cell suspension cultures

Two phenolic acids isolated from colourless cell cultures of Lithospermum erythrorhizon were identified as rosmarinic acid and lithospermic acid. However, these compounds were not detected in pigmented cell cultures producing shikonin derivatives.     

Effect of nutritional factors on shikonin derivative formation in Lithospermum callus cultures

Some nutritional factors affecting the biosynthesis of shikonin derivatives in callus cultures of Lithospermum erythrorhizon were examined. High sucrose concentrations increased the content of shikonin derivatives, but neither glucose nor fructose was effective for shikonin derivative formation. High concentrations of nitrogen sources inhibited or retarded shikonin derivative formation and streptomycin sulphate stimulated their biosynthesis. Addition of ascorbic acid increased the content of shikonin derivatives. Among some precursors tested only l-phenylalanine had a positive effect. At high concentrations, Ca²⁺ and Fe²⁺ inhibited the biosynthesis of shikonin derivatives.     

Shikonin production and secretion by hairy root cultures of Lithospermum erythrorhizon

Summary Hairy root cultures of Lithospermum erythrorhizon were established by transformation of in vitro grown shoots with Agrobacterium rhizogenes 15834. Hairy roots cultured on Murashige and Skoog solid medium did not produce any red pigments. However, the hairy roots cultured in Root Culture solid or liquid media produced a large amount of red pigments, which were released to the medium. The addition of adsorbents to the culture medium stimulated shikonin production by ca. 3-fold. Using this method an air-lift fermenter system was established, equipped with a XAD-2 column, which continuously produced ca. 5 mg/day of shikonin during a period of more than 220 days.     

Glucosylation of esculetin by plant cell suspension cultures

Cell suspension cultures of Lithospermum erythrorhizon, Gardenia jasminoides and Nicotiana tabacum were capable of glucosylating esculetin to esculin (7-hydroxycoumarin-6-O--D-glucoside). Especially, a culture strain of Lithospermum erythrorhizon was superior in the esculetin glucosylating capability; 40 to 50% of esculetin administered to the culture medium at early stationary growth stage was converted into esculin within 24 h. The rate of glucosylation was also dependent on the growth stage and the medium composition especially growth hormones and sugar.     

Cold storage of in vitro cultures of hybrid poplar shoots (Populus alba L. x P. grandidentata Michx.)

Shoot cultures of P. alba x P. grandidentata Crandon were maintained for more than 5 years at 4°C with minimal growth. The highest survival after 2 years and 5 years of cold storage were 70% and 25% respectively using 1-month of pre-storage culture on MS medium containing 1.33 M BA. When 5-year-old cold-stored shoot cultures were transferred to the greenhouse, color variations were observed. The frequencies of albino and red pigmented plants were 0.25% and 12.8%, respectively. A rosette type growth pattern was also observed on 0.3% of the long-term cold-stored plants. During long-term cold storage there were local disruption of cambium connections and the accumulations of chemicals in some cells, as observed by light microscopy.     

In Vitro Preservation of Asparagus Officinalis

A simple systems for in vitro storage of health asparagus germplasm was developed. High percent (90 %) of shoots cultured in a standard multiplication medium were maintained viable in vitro at 5 °C in darkness for 12 months. This percent was decreased to 60 % when cultures were stored for 18 months. At normal temperature, shoots and callus cultures also survived for 1 year under osmotic stress on medium containing 40 g dm^-3 mannitol.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of mature <Emphasis Type="Italic">Pinus sylvestris</Emphasis> buds stored at freezing temperatures

Changes of morphogenic competence in mature P. sylvestris L. buds due to frozen storage were investigated. The highest callus formation was registered on explants stored at –18°C for three months, but on explants stored for five months, it was also higher than in the control. Budding and development of needles in vitro was observed only for buds frozen three to five months. Peroxidase activity was lowest in these buds. In contrast, polyphenol oxidase activity in bud tissues continually increased during frozen storage. Within 10 months of frozen storage the content of starch and sugars in resting buds changed. It may be concluded that changes in composition of non-structural sugars in pine buds after five months of frozen storage are part of metabolic changes leading to loss of morphogenic capacity.     

Increased shikonin production by hairy roots of Lithospermum erythrorhizon in two phase bubble column reactor

Summary Plant hairy root cultures of Lithospermum erythrorhizon were carried out to produce shikonin derivatives by employing in situ extraction with n-hexadecane in a shake flask and a bubble column bioreactor. Over 95 % shikonin produced was recovered in the n-hexadecane layer. In flask cultures the maximum concentration of shikonin with n-hexadecane extraction was 3 times higher than that obtained without extraction. In the two phase bubble column reactor, 572.6 mg/L of shikonin and 15.6 g/L of dry cell mass were obtained after 54 days. Shikonin was produced at a constant level of 10.6 mg/L day during this period.