Production of polysaccharides by Silene vulgaris callus culture depending on carbohydrates of the medium

Sources of carbohydrate nutrition such as sucrose, glucose, and galactose, with the exception of arabinose, were shown to influence positively callus growth and polysaccharide (pectin silenan and acidic arabinogalactan) biosynthesis. Galactose was found to cause a stimulatory effect on yield and productivity of arabinogalactan. Low concentrations of sucrose failed to support the cell growth and polysaccharide biosynthesis. Increasing sucrose concentrations led to biomass accumulation but failed to enhance efficiency of the substrate utilization. The optimal medium for the campion cell culture growth was found to be one containing 30 g/liter of sucrose or a mixture of sucrose with glucose (in 15 g/liter). Increasing sucrose concentrations in the medium from 30 to 100 g/liter failed to significantly influence the polysaccharide yields while the polysaccharide productivity per liter of the medium grew due to promotion of culture productivity in biomass. Variations of the carbon sources in the nutrient media were shown to influence insignificantly the biochemical characteristics of arabinogalactan and silenan while an increase in the sucrose concentration to 50-100 g/liter led to a diminution of the galacturonic acid content in silenan and to changes in contents of the neutral monosaccharide residues in silenan and arabinogalactan.     

NUTRITIONAL REQUIREMENTS OF FRAXINUS CALLUS CULTURES

A satisfactory synthetic medium has been developed for continuous growth of Fraxinus pennsylvanica Marsh. callus cultures. The medium contains modified Reinert and White (1956) inorganic nutrient solution with 5 mg/liter Fe as NaFe-EDTA and supplemented with myoinositol 10 mg/liter, pyridoxine HCl 0.1 mg/liter, 2,4-dichlorophenoxyacetic acid 0.04 mg/liter, kinetin 1 mg/liter, sucrose 20 g/liter, and agar 10 g/liter. myo-Inositol, pyridoxine and an auxin are essential. α-Naphthaleneacetic acid is an effective alternate auxin. Kinetin and to some extent gibberellic acid improve the yields. Thiamine has no effect.     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.     

Accumulation of Glutamine by Suspension Cultures of Symphytum officinale

A callus was induced from the veins of a leaf of Symphytum officinale, comfrey, on a medium containing the inorganic elements reported by Murashige and Skoog with addition of 3% sucrose, 0.5 mg/liter 2,4-D and 0.3~3.0 mg/liter kinetin.

Suspension cultures of this cell line obtained from the callus were shown to accumulate a large amount of L-glutamine intracellularly, The effect of growth hormones and nutrients on accumulation of the amino acid has been examined in suspension cultures. The most suitable concentrations of 2,4-D and kinetin for glutamine accumulation were 0.3 mg/liter each. The presence of potassium nitrate as a nitrogen source was beneficial for growth and ammonium nitrate stimulated the accumulation of glutamine. High levels of these nitrogen sources in the medium were required for obtaining a high level of glutamine. The concentration of glutamine accumulated reached to approximately 20% of dry cell weight when S. officinale was incubated in the medium containing 0.495 % of ammonium nitrate and 0.570% of potassium nitrate which corresponded to three times higher levels than those in a Murashige and Skoog’s medium.

Most of the amino acid was found intracellularly but a small amount was excreted into the medium in the later stages of the incubation. Addition of a cationic surfactant, cetyltrimethylammonium bromide, to the cultures caused to increase the amount of the amino acid in the culture filtrate.

The contents of free amino acids in leaves of S. officinale were compared with those in the callus. The level of glutamine in the callus was 260 times higher than that in the intact plant.     

Theanine production by coupled fermentation with energy transfer using gamma-glutamylmethylamide synthetase of Methylovorus mays No. 9

Gamma-glutamylmetylamide synthetase (GMAS) of Methylovorus mays No. 9, produced by Eschericia coli AD494 (DE3) harboring pET21aGM, formed theanine from glutamic acid and ethylamine with coupling of the reaction with sugar fermentation of baker's yeast cells as an ATP-regeneration system. Theanine formation was stimulated by the addition of Mn(2+) to the reaction mixture, whereas Mg(2+) was less effective. Increases to a certain level in the concentrations of GMAS and the substrates in the mixture were effective in increasing theanine formation, but high concentrations of ethylamine (900 mM or more) inhibited yeast sugar fermentation, and eventually decreased theanine formation. The inhibitory effect of ethylamine was restored by increasing the concentration of potassium phosphate buffer in the mixture. Approximately 600 mM (110 mg/ml) theanine was formed in 48 h in an improved reaction mixture containing 600 mM sodium glutamate, 600 mM ethylamine.HCl, 300 mM glucose, 200 mM potassium phosphate buffer (pH 7.0), 30 mM MgCl(2), 5 mM MnCl(2), 5 mM AMP, 30 units/ml of GMAS, and 40 mg/ml of yeast cells. The yield of theanine was 100% on the substrates (glutamic acid and ethylamine) and also on the energy source (glucose consumed).     

Asiaticoside production from centella (Centella asiatica L. Urban) cell culture

Petiole explants of centella plants (Centella asiatica L. Urban) were cultured on Murashige and Skoog (MS) solid medium containing 20 g/L sucrose, supplemented with 1.0 mg/L benzylaminopurine and 1.0 mg/L naphthaleneacetic acid for callus production. To establish a cell suspension culture, 2 g of fresh callus was cultured in 50 mL of the same medium but without solid agent at a 100 rpm agitation speed. Every 2 g of culture was subcultured in fresh MS liquid medium for maintenance. After 24 days of culture at a 120 rpm agitation speed, the centella cell biomass reached a maximum of 9.03 g/50 mL on the same MS medium with 30 g/L sucrose and a 3 g inoculum size. A high performance liquid chromatography analysis showed that asiaticoside content in 24-day old suspension cultured cells (45.35 mg/g dry weight) was significantly higher (4.5 fold) than that of in planta leaves (10.55 mg/g dry weight).     

Regeneration of garlic plants (allium sativum L., CV. “Chonan”) via cell culture in liquid medium

Summary Aiming at the genetic improvement of garlic cultivars, a cell suspension protocol was established which includes the induction of friable callus, establishment of cells in liquid medium, plating, regeneration, and bulb formation. Calluses of various textures from compact to friable and from green to yellowish were obtained by culturing explants excised from inner leaves of garlic bulbs on Marashig-Shoog (MS) medium with 2,4 dichlorophenoxy acetic acid (2,4-D), (1.1 mg/liter [5.0 μM]), picloram (1.2 mg/liter [5.0 μM]), and kinetin (2.1 mg/liter [10 μM]). Friable callus occurred on MS-A contained 2,4-D alone (1.0 mg/liter [4.52 μM]) and this callus was used to develop cell suspension cultures, which were maintained in liquid MS-B medium with a 2,4-D/benzyl adenine (BA) (0.5 mg/liter [2.25 μM]: 0.5 mg/liter [2.22 μM]) ratio. High plating efficiency was obtained on MS-C medium with different naphthalene acetic acid/BA combinations. Regeneration occurred after transfer of the caulogenic mass to MS-C medium containing 10 mg/liter (74.02 μM) and 20 mg/liter (148.04 μM) adenine for 60 days, followed by transfer to adenine-free medium. Plantlets transplanted to soil showed normal phenology. Shoots grown on modified MS medium supplemented with indolylbutryic acid (3.0 mg/liter [14.7 μM]) stimulated bulb formation by 30 days in culture.     

Plant regeneration from isolated cells ofLavandula latifolia medicus

Summary Single cells were obtained from hypocotyl-derived callus ofLavandula latifolia Medicus. Cells were plated in Murashige and Skoog medium supplemented with indoleacetic acid (IAA), benzyladenine (BA), and several IAA-BA combinations. Cell division required the simultaneous presence of IAA and BA in the culture medium, but callus formation was only achieved with 0.1 or 1 mg/liter IAA and 2 mg/liter BA. To induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on the composition of both the callus induction and the shoot regeneration media, best results being obtained when calli grown in 1 mg/liter IAA and 2 mg/liter BA were subcultured to media containing 2 mg/liter BA and 15% coconut milk. Under these conditions, up to 75% of calli formed shoots that subsequently were rooted and established in soil.     

ATTEMPTS TO OBTAIN BACTERIA-FREE PLANTS OF PSYCHOTRIA PUNCTATA (RUBIACEAE): GROWTH AND ROOT FORMATION IN CALLUS CULTURES

Attempts were made to obtain bacteria-free plants of Psychotria punctata from tissue cultures. Stem explants and callus derived from them were induced to form roots but failed to form buds on Linsmaier and Skoog medium and 96 chemical modifications of it, including most of those known to induce bud formation in other species. Roots formed with ample IAA (2 mg/liter or more) and a low kinetin concentration (0.25 or 0.50 mg/liter). Adenine inhibited root formation in these media, but tyrosine did not. Tyrosine did lower the percentage of calluses commencing growth. When enzyme-hydrolyzed lactalbumin (1.3 g/liter), kinetin (0.5 mg/liter) and IAA (5 mg/liter) were added to Linsmaier and Skoog medium modified by decreasing inorganic nitrogen and increasing inorganic phosphate, callus grew at the fastest rate observed (increasing threefold in fresh weight in three weeks) and formed numerous roots. This was adopted as the stock callus medium. Casein hydrolysates also stimulated growth but less so than lactalbumin hydrolysate. When lactalbumin hydrolysate or a casein hydrolysate lacking tryptophan was supplied, growth occurred without added auxin if sufficient cytokinin was added. Cytokinin was required at unusually high concentration and was tolerated at still higher concentration. Formation, elongation, and branching of roots persisted on a saturated solution of BA which inhibited callus growth about 70 % and delayed callus senescence. Light caused earlier callus senescence after growth had ceased but did not affect callus growth or root formation. Light-induced senescence was prevented by a high cytokinin concentration.     

碳源对早花百子莲愈伤组织诱导及其增殖的生理特性影响

为揭示碳源对早花百子莲愈伤组织诱导与增殖的影响机理,该研究以早花百子莲的小花梗为外植体,比较分析30.0 g/L蔗糖、葡萄糖、麦芽糖在愈伤组织诱导、增殖中的效果,测定不同碳源种类处理下愈伤组织增殖相关生理特性,并根据细胞增殖效果、生理指标相关性进行优化验证。结果表明:(1)蔗糖、葡萄糖和麦芽糖碳源处理下,愈伤组织诱导率分别为86.00%、72.00%和59.67%,蔗糖碳源的愈伤组织诱导率比葡萄糖和麦芽糖分别显著提高19.44%和44.13%(P<0.05),蔗糖碳源较葡萄糖和麦芽糖碳源的愈伤组织大小分别显著增加22.44%和90.09%(P<0.05);愈伤组织增殖阶段,蔗糖碳源能够同时维持良好的细胞增殖效率及活性,而葡萄糖碳源的愈伤组织增殖快、状态差,麦芽糖处理增殖慢、状态佳;蔗糖转换葡萄糖碳源后愈伤组织细胞团大小、细胞活性明显下降;蔗糖转换蔗糖、蔗糖转换麦芽糖的效果较好。(2)培养基碳源显著调节愈伤组织增殖阶段的糖代谢、内源激素代谢和氧化胁迫平衡。(3)愈伤组织的主要糖组分为淀粉、葡萄糖;淀粉、麦芽糖含量与细胞团大小相关性高,以蔗糖为碳源的培养基中添加麦芽糖,愈伤组织增殖效果良好,细胞团颜色鲜黄,活性较强。(4)愈伤组织的结合态IAA、GA4、CTK含量与细胞团大小具有一定的相关性,培养基中添加1.0 mg/L 6-BA可显著促进愈伤组织增殖效率(P<0.05)。(5)ROS活性与POD、CAT活性以及POD活性与H2O2含量均呈显著负相关关系(P<0.05),而POD与CAT活性呈极显著正相关关系(P<0.01)。(6)验证及优化实验结果表明,增殖培养基中添加麦芽糖、6-BA可有效促进早花百子莲愈伤组织继代增殖效果,其中麦芽糖可以保持、改善细胞活性,而6-BA主要促进了细胞增殖。研究发现,蔗糖为早花百子莲愈伤组织诱导及增殖的最佳碳源,蔗糖、麦芽糖组合碳源利于愈伤组织细胞活力维持,而毒莠定(PIC)与6-BA组合利于愈伤组织细胞增殖,最佳增殖培养基为:MS+0.5 mg/L PIC+1.5 mg/L 6-BA+15.0 g/L蔗糖+15.0 g/L麦芽糖+7.0 g/L琼脂。     

生姜离体叶片愈伤组织悬浮细胞系建立与植株再生

以‘莱芜大姜’为试材,研究了生姜离体叶片愈伤组织的诱导以及细胞悬浮系建立与植株再生。结果表明,以生姜试管苗叶片为外植体,接种到MS+1.0 mg/L 2,4-D+0.5 mg/L 6-BA+30 g/L蔗糖的培养基上,可有效诱导出生长迅速、质地疏松的愈伤组织。将获得的愈伤组织接种到MS+0.15 mg/L 2,4-D+6.0 mg/L 6-BA+30 g/L蔗糖的液体培养基上,25℃黑暗条件下震荡培养25-30 d,可建立分散性好、生长迅速的悬浮细胞系,细胞悬浮系培养的适宜参数为:初始接种量为1.0-1.5 g,继代培养的适宜间隔期为15 d,继代培养液体培养基更新比例为3/4。将悬浮细胞接种到固体培养基MS+0.2 mg/L NAA+10.0 mg/L 6-BA+30 g/L蔗糖上可获得再生植株。     

The Influence of Different Factors on Cell Growth and α-tocopherol Content of Culture from Carthamus tinctorius

Calli were induced from root, hypocotyl, cotyledon and flower bud of Carthamtus tinctorius. All calli had the capability to synthesize α-tocopherol. Among these calli, the hvpocotylcallus was better than others in cell growth rate and α-toeopherol content. Culture conditions could intensively influence the growth rate and α-tocopherol production of callus from Carthamus tinctorius. Sucrose (30g/L) was good for callus growth and glucose (30g/L) was good for α-tocopherol accumulation. High concentration (0.55%) of inositol could obviously stimulate both growth rate and α-tocopherol synthesis of callus. The inoculum quantity for best callus growth was 0.035 to 0.067g dry wt/flask (50ml volume). In addition, α-tocopherol content was effectively increased by culture callus in high CO2 concentration. Studies on optimum cuhure conditions of callus culture showed that the callus growth rate, α-tocopherol content and yield were 1.88, 2.03 and 3.30 times respectively as high as of the control by administration of 0.45%–0.55% inositol, 10% coconut milk, 0.1–0.5% casamino acid, 30g/L sucrose and 10g/L glucose.     

Callus induction and maintenance of Zea mays kernels

Summary Totipotent tissue cultures of maize (Zea mays L.) have previously been initiated from various explant tissues. In this paper, we present an alternative source of callus induction.A callus of maize (G 204 hybrid) was obtained from intact kernels grown on Linsmair and Skoog RM medium supplemented with 20 mg 2,4-dichlorphenoxyacetic acid (2,4-D) per litre. The callus growth was greatest from the first node of the seedling shoot. Occasionally, callus growth was observed from the radicle and coleopticle regions. The callus was easily transferred and maintained on a medium with 2 mg/L 2,4-D. This callus formed numerous roots and leaf-like structures when grown on a medium containing 800 mg/L yeast extract, 30 g/L sucrose and 10 g/L agar.     

Establishment of an in vitro regeneration system for genetic transformation of selected sugarcane genotypes

A good culture system provides considerable quantities of highly regenerable target tissues. Embryogenic callus cultures are ideal for micro-projectile-mediated transformation, because regenerable cells are not very stable. Effective exploitation of genetic transformation requires good regeneration systems. We selected three sugarcane genotypes for the establishment and optimization of good in vitro regeneration systems, viz., S-2003-us-359, S-2006-sp-30, and S-2003-us-165. Three callus induction media were investigated. These media were composed of Murashige and Skoog (MS) medium salt plus 1, 2, and 3 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Medium with 3 mg/L 2,4-D gave the greatest mass of embryogenic calli. The calli produced on the three callus induction media were transferred to 18 types of regeneration media (RM1-RM18). They varied with respect to plant growth regulators and sucrose levels but the basal medium was MS. Two levels of sucrose (30 and 40 g/L), three levels of 2,4-D (0.1, 0.25, 0.5 mg/L) and three levels of 6-benzylaminopurine (0, 0.25 and 0.5 mg/L) were studied in the regeneration media. The effects of callus age on regeneration were evaluated by transferring the calli to regeneration media after 15, 21, 28, and 35 days of culture. The 21-day-old callus of the genotype S-2003-us-359 on RM3 yielded the largest number of plants and was selected as the best for transformation. Six RAPD DNA primers were used to check genetic stability; this medium did not affect the sugarcane genomes.     

小麦成熟胚愈伤组织诱导及分化研究

以2个小麦品种成熟胚为外植体进行离体培养,研究了不同预处理、不同2,4-D浓度及与KT组合、不同蔗糖浓度等因素对愈伤组织诱导及分化的影响。结果表明:4℃低温预处理可提高愈伤组织的出愈率及再生苗率,2个材料的出愈率及再生苗率均达到90%和30%以上;在不同预处理条件下,2,4-D浓度对出愈率及再生苗率的影响与基因型有关,2,4-D浓度为1~2 mg/L更有利于愈伤组织诱导及分化;附加KT能缓解高浓度2,4-D对再生苗率的抑制作用,而对于在1、2 mg/L 2,4-D的培养基中附加KT则不表现这种作用;蔗糖浓度则在30 g/L条件下更有利于愈伤组织诱导。因此通过4℃低温预处理,在MS基本培养基中附加1~2mg/L 2,4-D及30 g/L蔗糖亦可促进小麦成熟胚愈伤组织的诱导和分化。     

An efficient and reproducible method for regeneration of whole plants from mature seeds of a high yielding Indica rice (Oryza sativa L.) variety PAU 201

Tissue culture is one of the tools necessary for genetic engineering and many other breeding programs. Moreover, selection of high regenerating rice varieties is a pre-requisite for success in rice biotechnology. In this report we established a reproducible plant regeneration system through somatic embryogenesis. The explants used for regeneration were embryogenic calli derived from mature seeds cultured on callus induction media. For callus induction mature seeds were cultured on MS medium containing 30 g/l sucrose combined with 560 mg/l proline and 1.5-3.5 mg/l 2,4-D and 0.5-1.5 mg/l Kin. For plant regeneration, embryogenic calli were transferred to MS medium containing 30 g/l sucrose, supplemented with 1.0-3.0 mg/l BAP, 0.5-1.5 mg/l Kin and 0.5-1.5 mg/l NAA. The highest frequency of callus induction (44.4%) was observed on the MS medium supplemented with 2.5 mg/l 2,4-D, 0.5 mg/l Kin, 560 mg/l proline and 30 g/l sucrose. The highest frequency of shoot regeneration (42.5%) was observed on the MS medium supplemented with 2.0 mg/l BAP, 0.5 mg/l NAA and 0.5 mg/l Kin. The plantlets were hardened and transferred to soil in earthen pots. The developed method was highly reproducible. The in vitro developed plants showed normal growth and flowering under glasshouse conditions.     

Effects of N-1-naphthylphthalamic acid on the growth and bud formation of tobacco callus grown in vitro

The effect of N-1 -naphthylphthalamic acid (NPA), indole-3-aceticacid (IAA) and kinetin on callus growth and bud formation wasstudied mainly by a tobacco callus culture method. Callus producedfrom Nicotiana tabacum var. Wisconsin 38 was used as the testplant material. Callus growth on nutrient agar containing 2mg/liter of IAA was promoted by NPA added at a concentrationof 0.5 mg/liter with 0.4 mg/liter of kinetin or by NPA addedat 5 mg/liter in the absence of kinetin. At a high concentrationof 50 mg/liter, however, NPA inhibited growth on the mediumcontaining 2 mg/liter IAA and no kinetin. Kinetin reduced thisNPA inhibition. In the presence of 0.4 mg/liter kinetin and2 mg/liter IAA, when the concentration of NPA was 50 mg/liter,buds were initiated after calluses were grown on the test mediumfor 7 weeks in dim light, but no buds formed when NPA was omittedfrom the above medium. The control of callus growth and bud initiation is based onthe active ratio of auxin (IAA) to cytokinin (kinetin) in themedium and NPA added to the medium can promote or inhibit callusgrowth and induce bud formation. Therefore, it is proposed thatNPA can itself reduce auxin activity or enhance cytokinin activityand hence change the active ratio of the two regulators. NPAmay enhance the activity of cytokinin (here supplied as kinetin)but cannot substitute for it. ¹Present address: Department of Biology, Wisconsin State University,Oshkosh, Wisconsin 54901, U. S. A. (Received March 10, 1969; )     

不同培养条件对悬浮培养茶叶细胞生长及茶氨酸合成的影响*

婺源绿茶嫩叶用MS 培养基( 加IBA 2 mgPL, 6-BA 4 mgPL) 进行茶叶愈伤组织悬浮培养, 研究了不同培养条件对茶叶细胞悬浮培养过程中细胞生长与茶氨酸合成的影响。结果显示, NH4+PNO3- 110P6010 mmolPL、K+ 10010 mmolPL、Mg2+ 310mmolPL、H2PO4- 310 mmolPL、蔗糖3010 gPL、水解酪蛋白210 gPL 条件下, 茶叶细胞生长量和茶氨酸积累量均达到最高值; 提高培养基中蔗糖和水解酪蛋白浓度可使细胞对数生长期和稳定期得到延长, 从而有利于茶氨酸积累; H2 PO4- 浓度主要影响细胞生长速率和茶氨酸积累速率的同步性, 低H2 PO4- 浓度环境中茶氨酸积累速率峰值滞后于细胞增长速率峰值, 高H2PO4- 浓度环境中早于细胞生长速率峰值出现时间; K+ 和Mg2+ 对细胞生长的影响不明显, 但影响细胞茶氨酸合成酶活性, 维持适量的K+和Mg2+ 有利于茶氨酸积累。添加盐酸乙胺可大幅度提高茶氨酸积累量, 并且先加入一定量盐酸乙胺再每天进行少量补充, 茶氨酸合成量比一次性加入的效果要好。茶叶细胞生长和茶氨酸积累高峰期在整个培养过程的第19~ 22 天出现, 从生产效率考虑, 培养周期以19~ 22 天为宜。     

Rosmarinic acid synthesis in transformed callus culture of Coleus blumei benth

Agrobacteria mediated Coleus blumei tumour tissues were cultured in vitro on MS medium. Sixteen diversified transformed callus cultures were maintained for several years in the absence of plant growth regulators and antibiotics without affecting the growth rate. Rosmarinic acid was detected spectrophotometrically in all tissue lines but in different quantities. The highest rosmarinic acid accumulation detected was 11% of dry tissue mass. The relation between culture growth and rosmarinic acid production was investigated in three callus lines. The lines showed different rosmarinic acid accumulation in relation to their growth rate; it was either parallel or inversely related to the tissue growth. The effects of certain medium constituents on the callus growth and rosmarinic acid accumulation were examined in four tumour cell lines. Addition of 4% or 5% sucrose stimulated rosmarinic acid synthesis and decreased callus growth. Nitrogen reduction to one half or one quarter of initial concentration did not affect rosmarinic acid synthesis and decreased callus growth in three lines, while it increased rosmarinic acid accumulation and callus growth in one line. Addition of 0.1 mg/l Phe stimulated rosmarinic acid production in two lines but had little effect on the rosmarinic acid level in others. Rosmarinic acid production was significantly improved on modified macronutrients, where the Ac2 line produced 16.5 mg of rosmarinic acid per tube (0.2 g of dry wt) after being in culture for 35 days.     

Intracellular concentrations and metabolism of carbon compounds in tobacco callus cultures: effects of light and auxin

Callus cultures derived from pith tissue of Nicotiana tabacum were grown on two media either under continuous illumination or in complete darkness. The first medium limited greening ability of callus grown in the light (3 milligrams per liter naphthalene acetic acid, 0.3 milligram per liter 2-isopentenylaminopurine, Murashige and Skoog salts, and 2% sucrose). The second medium encouraged chlorophyll synthesis (greening) though not shoot formation (0.3 milligram per liter naphthalene acetic acid; 0.3 milligrans per liter 2-isopentylaminopurine). To measure intracellular concentrations, calli were grown for 15 days on these standard media containing [U-¹⁴C]sucrose. The dry weight proportions of the calli (as a fraction of fresh weight) and many metabolite concentrations nearly doubled in light-grown cells compared to dark-grown cells and increased 30 to 40% on low-auxin media relative to high-auxin media. Glutamine concentrations (from 4 to 26 millimolar) were very high, probably due to the NH₃ content of the media. Proline concentrations were 20-fold higher in calli grown on low-auxin media in the light (green cells), possibly a stress response to high osmotic potentials in these cells. To analyze sucrose metabolism, callus cells were allowed to take up 0.2% (weight per volume) [U-¹⁴C]sucrose for up to 90 minutes. In callus tissues and in pith sections from stems of tobacco plants, sucrose was primarily metabolized through invertase activity, producing equal amounts of labeled glucose and fructose. Respiration of ¹⁴CO₂ followed the labeling patterns of tricarboxylic acid cycle intermediates. Photorespiration activity was low.