In vitro propagation ofCereus peruvianus mill. (cactaceae)

Summary Cereus peruvianus seedlings were used as a source of stem explants to determine the effective conditions for inducing and maintaining callus tissues in a state of rapid growth, as well as to obtain plants regenerated from callus cultures. Factorial combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin in MS medium were tested, and we concluded that the 18.1µM 2,4-D and 18.6 or 27.9µM kinetin combinations were suitable for callus induction. The cactus shoots were produced from the friable callus; root elongation occurred within 2 wk in medium without 2,4-D and with 18.6µM kinetin. This method can be used to rapidly produce manyC. peruvianus plants.     

Histological study of callus formation and root regeneration from mung bean (Vigna radiata W.)

We have established a reproducible culture system for callus formation and root development from juvenile stem segments of mung bean(Vigna radiata). In particular, we have studied the influence of plant growth regulators. Induction of calli from young stem explants was very effective on MS inorganic salts supplemented with 0.5 mg/L 2,4-D and 1.0 mg/L kinetin. In regenerating adventitious roots from callus tissues, we found that a combination of 0.75 mg/L NAA, 1.5 mg/L kinetin, and MS salts resulted in 20% efficiency. Histological examination showed that callus tissues originated from out-growths of the cambium rings through de-novo meristematic activity. Those rings were localized outside the vascular cambium. Adventitious roots that developed from root primordia originated from the center of the Callus masses. These primordia produced tracheid-like cells, which then became meristemoid cells for the cambium. Newly formed adventitious roots had the typical tetrarche actinostele type.     

Some Accounts on Culture Conditions of Callus Tissues of Solanum laciniatum Ait. for Producing Solasodine

Production of solasodine in callus cultures of Solanum laciniatum Ait. was examined under several culture conditions. The steroidal alkaloid was produced more actively in rapidly proliferating callus tissues cultured on PN medium. The alkaloid concentration in the tissue was about 0.05% (dry weight basis) during the first 5 weeks’ culture. The highest accumulation of the alkaloid per culture was obtained with 2,4-d concentration in the medium at 1~2 ppm. It is noteworthy that the alkaloid production was not inhibited by such high concentration of 2,4-d as up to 10 ppm in the medium. Supplementation of kinetin slightly increased the alkaloid production.     

Growth and tropane alkaloid production inAgrobacterium transformed roots and derived callus ofDatura

Small callus pieces excised from theAgrobacterium transformed root line D₂ ofDatura stramonium, were cultured onto solidified MS medium supplemented with a 1.0 μM kinetin and three different concentrations (0.1, 0.5 and 1.0 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D), and were examined for their alkaloid productivity in relation to organization level and growth rate. Growth of transformed roots (in a MS liquid medium without plant growth regulators) was greater than that of transformed calli excised from them and cultured separately. The addition of 1.0 μM 2,4-D to the culture medium had a positive effect on callus biomass production, while it inhibited root formation by this tissue (the lower the 2,4-D concentration in the medium the greater the number of roots which emerged from the calli). Hyoscyamine production was also higher in the transformed roots than in the transformed calli, and in these tissues the production of hyoscyamine was positively correlated with organogenesis index (i.e. its ability for rooting). At the same time, the epoxidation of hyoscyamine to scopolamine only took place in the transformed calli. This occurred to a greater extent at the lower concentrations of 2,4-D in the culture medium. The mode through which the 2,4-D could control the alkaloid production of transformed callus is discussed.     

Production of 8-epi-tomentosin by plant cell culture ofXanthium strumarium

This study was conducted to establish a plant cell culture system for the production of medically important secondary metabolites fromXanthium strumarium. The effects of plant growth regulators including NAA, 2,4-D, kinetin, and ABA were examined in terms of callus induction, maintenance of callus and suspension cultures. It was shown that callus was induced upon treatment with NAA while embryo was induced after treatment with 2,4-D. Callus formation was further improved by treatment with ABA and NAA. The level of callusing increased by 17–29% for the seed case, cotyledon, leaf, and hypocotyl and by 96% in the case of the root. Suspension cell lines were established using calli produced from cotyledon, hypocotyl and root and cultured at 25°C under light conditions. The cells grew up to 15 g/L with NAA 2 ppm, BA 2 ppm, and ABA 1 ppm treatment. Supernatants of suspension cultures of cell lines derived from coyledon and hypocotyl produced some distinctive secondary metabolites, one of which was identified as 8-epi-tomentosin, which belongs to the xanthanolides. The amounts of 8-epi-tomentosin produced by the cotyledon-and hypocotylderived cell lines were 13.4 mg/L and 11.0 mg/L, respectively.     

SOMATIC EMBRYOGENESIS AND ORGANOGENESIS FROM CALLUS CULTURES OF SOLANUM CAROLINENSE

Culture of stem segments of Solanum carolinense L. on medium supplemented with 10 mg/1 2,4-dichlorophenoxyacetic acid and 1 mg/1 kinetin, induced callus formation. When subcultured on medium lacking 2,4-D but containing a cytokinin, the callus regenerated. The mode of regeneration depended on the type and concentration of cytokinin employed; high concentrations of benzyladenine and all concentrations of kinetin promoted organogenesis, while low concentrations of benzyladenine induced somatic embryogenesis in addition to organogenesis. With age and continued subculture on 2,4-D containing medium, callus progressively lost its ability to regenerate when the auxin was replaced by cytokinin. In conjunction with previous studies on regeneration from anther cultures of S. carolinense, it appears that in both cases, 2,4-D is required for callus initiation and proliferation but must be exchanged for a cytokinin before differentiation will occur. However, since it was not possible to induce embryogenesis in pollen-derived callus, developmental potential may be influenced by the ploidy level of responding cells in culture.     

Alcohol dehydrogenase (EC 1.1.1.1) isozymes as markers at 2,4-dichlorophenoxyacetic acid × kinetin combinations in callus cultures ofCereus peruvianus (Cactaceae)

Alcohol dehydrogenase (ADH; EC 1.1.1.1) isozymes were investigated in tissue ofCereus peruvianus cultured in different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin. Five ADH isozymes were detected in starch gel and showed different patterns in seeds, seedlings, calli cultured at 32 and 22°C, and plants regenerated from calli cultured in three 2,4-D and kinetin combinations. Four phenotypes formed by different combinations of ADH-2, ADH-3, ADH-4, and ADH-5 were detected in calli cultured at 32°C and in plants regenerated from calli. ADH-1 isozyme was detected only in calli subcultured for 1 or 2 weeks at 22°C and was indicated as a marker of stress conditions that affect the growth ofC. peruvianus callus tissues in culture. ADH phenotypes with either a higher or a lower number of isozymes were detected in different proportions in the callus tissues cultured in media containing different 2,4-D and kinetin ratios. ADH isozyme patterns were found to be sensitive markers at the highest kinetin concentration or at high kinetin/2,4-D ratios. The results indicate a high correlation between the ADH isozyme patterns and the capacity for regeneration. Thus, ADH isozymes are indicated as good biochemical markers and as a powerful tool for monitoring studies ofC. peruvianus callus cultures.This research was supported by the CNPq.     

Toward the production of artemisinin through tissue culture: Determining nutrient-hormone combinations suitable for cell suspension cultures

Summary Artemisia annua L. is the source of a potent antimalarial, artemisinin. As part of a program to produce artemisinin through tissue culture, a series of 14 multifactorial experiments were conducted to determine suitable conditions for initiating and maintaining friable callus fromA. annua. In the first six experiments, three different nutrient formulations [Gamborg B5 (B5), Murashige and Skoog (MS), and Whetmore and Rier (WR)], each with 32 combinations of auxins and cytokinins [2,4-dichlorophenoxyacetic acid (2,4-D) with benzyladenine (BA), or 1-naphthaleneacetic acid (NAA) with 6-furfurylaminopurine (kinetin)], were tested. Both B5 and WR nutrients supported friable callus formation from leaf explants with some combinations of auxin and cytokinin. Inasmuch as friable callus seemed to be produced over a wider range of auxin and cytokinin concentrations in combination with B5, the remaining experiments were conducted solely with this nutrient formulation. In the remaining eight experiments, it was determined that friable callus formed when combinations of NAA with kinetin or 2,4-D and BA were used with B5 medium. Lighter colored, more friable callus formed in response to 2,4-D and BA than with NAA and kinetin. No single combination of concentrations of auxin and cytokinin seemed to be “ideal” for producing friable callus. Ranges of 2,4-D from 0.5 to 2.0 with BA between 0.025 and 0.1, or NAA between 0.5 and 2.0 with kinetin between 0.5 and 1.0 mg/liter, produced acceptable results.     

Induction of somatic embryogenesis and genetic transformation of ohio buckeye (Aesculus glabra willd.)

Summary Mature embryo axes of the Ohio buckeye were germinated on a medium containing 1 mg gibberellic acid (GA) per 1. Three wk following germination, stem, petiole, and leaf blade tissues were excised and placed on media containing either 1 mg (4.5 μM) 2,4-dichlorophenoxy acetic acid (2,4-D) per 1, 1 mg (4.7 μM) kinetin per 1, 1 mg of both 2,4-D (4.5 μM) and kinetin (4.7 μM per 1, or 2 mg of both 2,4-D (9.1 μM) and kinetin (9.3 μM) per 1. Embryogenic tissue was formed only from stem segments after 2–3 mo. of culture on media containing both 2,4-D and kinetin. Embryogenic tissue could be either maintained on solid medium for proliferation of embryogenic callus or placed in liquid medium for proliferation of embryogenic suspension cultures. For transformation of suspension cultures, tissues were inoculated with Agrobacterium EHA105 containing the binary plasmid Vec035, briefly sonicated, and cultured in the presence of 100 μM acetosyringone for 2 d. To eliminate Agrobacterium, tissues were washed and placed in liquid proliferation medium containing either 500 mg Cefotaxime per 1 or 400 mg TimentinŖ per 1. Selection on 20 mg hygromycin per 1 was initiated 2 wk after inoculation, and after an additional 10 wk, hygromycin-resistant tissue was isolated and separately cultured. Although some hygromycinresistant clones were recovered with no sonication treatment, four to five times more clones were obtained following sonication. Putative transformed clones were confirmed to be transgenic via both histochemical β-glucuronidase (GUS) assay and southern hybridization analyses. Development of transgenic embryos occurred on a growth regulator-free medium containing 3% sucrose. After 2 mo. of embryo development, the embryos were transferred to fresh medium for germination.     

Proliferative activity of callus cultures of Taxus baccata L. in relation to anticancer diterpenoid taxol biosynthesis

Summary Stable growing callus cultures were recovered from stem segments of Taxus baccata. Callus developed from the peridermal cells. The content of taxol in callus tissues varied in the range of [0.0003–0.001% on dry weight basis] and was higher in fast growing calli compared to slow growing calli.Abbreviations BAP 6-benzylaminopurine; 2,4-D - 2,4-dichlorophenoxyacetic acid - KIN kinetin - NAA -naphthyl-1-acetic acid - PVP polyvinylpyrrolidone - SEM scanning electron microscopy     

Plant regeneration of Chorispora bungeana via somatic embryogenesis

Summary A method was developed for in vitro regeneration of plants via somatic embryogenesis in Chorispora bungeana, an alpine plant with freeze-tolerance, using cell suspensions initiated from leaf-derived callus. Primary calli were induced from leaves of C. bungeana grown on Murashige and Skoog (MS) media supplemented with 4.0 mg l⁻¹ gibberellic acid (GA₃), 0.2 mgl⁻¹ α-naphthaleneacetic acid (NAA) and 0.2 mgl⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension culture was initiated by incubating the callus particulates in liquid MS medium supplemented with 1.0 mgl⁻¹ kinetin (KT) and 0.2 mgl⁻¹ NAA. Individual early cotyledonary-stage somatic embryos isolated from cell suspension developed into whole plants on medium containing high levels of sucrose (60 and 90 gl⁻¹), whereas lower sucrose concentrations (0 and 30 gl⁻¹) were inhibitory to main root development. On the MS medium with 90 gl⁻¹ sucrose, one regenerated plant exhibited hetero-morphologic leaves, while other plants grown on different media showed a transformation from stem to root.     

Tissue culture inHaworthia

Effects of three auxins and kinetin on growth of the calluses of two species ofHaworthia, H. aristala andH. setata, were investigated. Stock calluses derived from the flower buds of these species were maintained for two years on RM-1964 agar medium containing 5 mg/l NAA. Small pieces of the stock calluses were transferred to the basal medium containing either auxin, IAA, NAA or 2,4-D in six different concentrations (0.1–50 mg/l) combined with three concentrations (0–2 mg/l) of kinetin; in total, 54 kinds of media were used. Fresh weight of the calluses was measured 0 to 30 days from transfer and transformed to the natural logarithm. The linearity of their growth curves against the culture period was tested. The growth curves of theH. aristata calluses grown in dark and under continuous light and that of theH. setata callus grown in dark gave similar regression coefficients of 0.07 to 0.11, indicating that the doubling time of the callus mass was about 6.3 to 10.1 days. After 42 to 50 days from inoculation, the fresh weight of each individual callus was recorded, and the data were statistically analyzed. All auxins at the concentration of 50 mg/l significantly inhibited callus growth. Kinetin did not affect growth of theH. aristata callus in dark, while its effect on theH. setata callus was detected under light. Interaction of kinetin was found with IAA and 2,4-D inH. aristata and with IAA and NAA inH. setata. REsponses of theH. aristata callus to auxins and kinetin, when grown in dark, were different in several points from those of theH. setata callus grown under light. The best callus growth was observed in the following media; 0.2 mg/l kinetin supplemented with 1 mg/l IAA, or 0.5 mg/l 2,4-D, and 2 mg/l kinetin with 0.5 mg/l NAA inH. aristata, and 0.2 mg/l kinetin supplemented with 1 mg/l IAA, 5 mg/l NAA or 0.1 mg/l 2,4-D inH. setata. Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 413.     

Chemical Regulation of Callus Growth, Organogenesis, Plant Regeneration, and Somatic Embryogenesis in Antirrhinum majus Tissue and Cell Cultures

Excised stem explants of Antirrhinum majus L. var. ‘Kymosyblanc’ were grown in a denned medium to investigate factorsinfluencing bud and root development, callus induction, andsomatic embryogenesis. Auxins such as indoleacetic acid (IAA)and naphthaleneacetie acid (NAA) caused limited callus developmentand abundant root formation, whereas 2,4-dichlorophenoxyaceticacid (2,4-D) promoted soft friable callus with embryos and occasionaldevelopment of thick abnormal roots. 2-Naphthoxyacetic acid(NOA) and coconut milk (CM) used together induced friable greencallus growth and differentiation of small globular embryoswhich eventually developed into plantlets after transfer toauxin-free agar mineral medium containing sucrose. Cytokininssuch as benzyladenine (BA), zeatin, and kinetin induced compactgreen callus but in the absence of auxin failed to promote organogenesis.The interaction of IAA and kinetin resulted in the regenerationof the whole plant from stem explants. When NAA was used withkinetin, shoot development was totally inhibited and abundantroots were formed. Thus, the alternative morphogenetic eventsprobably reflect the biochemical subtleties occurring withinthe callus as a result of differences of actual endogenous levelsof growth substances in the tissues studied. These experimentshave been performed and interpreted on a histological basis.     

The Initiation and Maintenance of Vicia faba Tissue Cultures

Explants obtained by removing the radicle tip and the plumule from embryos of Vicia faba have been induced to form callus in culture. Of a range of agar-solidified culture media tested, only that of Schenk and Hildebrandt (1972) was consistently successful. Improved growth, measured as increasing fresh weight was obtained by increasing the nitrogen content of the medium, either as potassium nitrate or as ammonium nitrate. A kinetin concentration of 0.01 mg/1 (5 × 10⁻⁸M) and a 2,4-dichlorophenoxyacetic acid (2,4-D) concentration of 0.5 mg/1 (2.3 × 10⁻⁶M) allowed optimum initial callus growth. A 2,4-D concentration of 2.3 × 10⁻⁸M, while insufficient to induce callus formation was able to inhibit lateral root development which occurred from embryo explants cultured without added 2,4-D. Subcultured tissue grew well on media supplemented with casein hydrolysate or a mixture of the eight most common amino acids in casein hydrolysate. Growth in subcultures was inhibited by two other amino acid mixtures used by other workers for different species.     

Effect of synthetic auxins on callus induction from tea stem tissue

A study was initiated to establish an in vitro culture protocol for tea (Camellia sinensis). Explant sources, disinfestation methods and culture media were examined. Segments (divots) were dissected from greenwood stem (current year growth) internodes of field grown plants. Disinfestation was achieved by separate treatments of 3.75% sodium hypochlorite and 7.5% CaCl₂. MS medium with sucrose (30 g/L), inositol (100 mg/L) and thiamine-HCl (1.3 mg/L) and kinetin was used with combinations of the auxins: (2,4-dichlorophenoxy) acetic acid (2,4-D), (2,4,5-trichlorophenoxy) acetic acid (2,4,5-T), (naphthalene) acetic acid (NAA) and 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (Picloram). Picloram (10^-7M) induced the most callus proliferation without kinetin. At a constant level of kinetin (10^-5M), the concentrations inducing the most callus growth were 10^-7M for 2,4-D, 10^-6M for 2,4,5-T, 10^-7M for Picloram and 10^-8M for NAA. A factorial test of 2,4,5-T and kinetin concentrations showed the optimum for callus growth was 10^-7M and 10^-5M, respectively.Technical Contribution No. 2532 of the South Carolina Agricultural Experiment Station, Clemson University.Graduate Research Assistant and Professor, respectively.     

Tissue culture inHaworthia

Effects of three different auxins and kinetin in various combinations on greening and redifferentiation of the callus ofHaworthia setata were investigated. All auxins at the concentration of 50 mg/l inhibited callus greening. NAA in combination with kinetin promoted both callus greening and production of redifferentiated shoots. Low concentrations of IAA without kinetin promoted redifferentiation of shoots, but not callus greening. Addition of 2,4-D completely inhibited both greening and redifferentiation regardless of the level of kinetin except for the effects on shoot formation in the medium with 0.1 mg/l 2,4-D added. The calluses with the highest chlorophyll content were observed in the medium containing 2.0 mg/l kinetin without any auxins or with 0.1 mg/l NAA added. Most frequent shoot redifferentiation was observed in the medium containing 0.1 mg/l IAA without kinetin (redifferentiation rate; 67%), followed by the medium containing 10 mg/l NAA with 2.0 mg/l kinetin (44%), and 0.1 mg/l 2,4-D with 0.2 mg/l kinetin (33%). Generally, higher degrees of greening were associated with better growth. However, the auxins (IAA, NAA and 2,4-D) given at concentrations optimal for growth did not exhibit the highest degree of callus greening. Differences of the three auxins in their actions and interaction with kinetin were disclosed. Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 423     

Genotypic variability for callus formation and plant regeneration in rice (Oryza sativa L.)

Summary Sixty rice varieties (Oryza sativa L.), belonging to three subspecies, japonica, indica and javanica (some japonicaXindica hybrids were included), were compared for their capacity for callus growth and plant regeneration. Tissue cultures initiated from mature seeds on Murashige and Skoog's (1962) medium with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) were transferred to a medium containing 0.02 mg/l 2,4-D and 10 mg/l kinetin, from which plantlets were regenerated. Large variabilities in callus growth and plant regeneration potentials were revealed among the varieties tested. Most japonica varieties formed a callus that weighed more than 100 mg per seed 30 days after inoculation, and showed a relatively high regenerative potential, whereas indica varieties, japonica-indica hybrids and javanica varieties showed poor callus growth and plant regeneration, although considerable varietal variation was observed in each subspecies. The callus growth potential was not correlated with the plant regeneration potential. Histological observations revealed that the epithelium cells of the scutellum mainly proliferated to form a callus, from which shoot and root primordia were differentiated independently from each other. The shoot primordia developed into plantlets when roots were formed adventitiously. In a few cases, shoots and roots were bilaterally initiated from a single primordium.     

Forskolin synthesis in in vitro cultures of Coleus forskohlii Briq transformed with Agrobacterium tumefaciens

Agrobacterium tumefaciens mediated tumor tissue and shooty teratomas of Coleus forskohlii were cultured in vitro. Forskolin was detected in tumorous callus (0.002%), rhizogenic callus (0.011%) and root cultures (0.014%), but not in shooty teratomas. Forskolin synthesis and accumulation in tumorous C. forskohlii cultures may permit the elucidation of diterpene metabolism in this species.Abbreviations B₅ medium after Gamborg et al. (1968) - 2,4-D 2,4, dichlorophenoxyaceticacid - MS medium after Murashige and Skoog (1962) - BAP benzylaminopurine - IBA indole-3-butyric acid - Kn kinetin - CH casein hydrolysate - T-DNA transferred DNA     

Differential alcohol dehydrogenase and malate dehydrogenase isozyme expression in long-term callus tissue cultures ofCereus peruvianus (Cactaceae)

Alcohol dehydrogenase (ADH) and mitochondrial malate dehydrogenase (mMDH) isozymes were tested as markers to study the effect of a high kinetin concentration on isozyme phenotypes and on the development ofCereus peruvianus callus tissue culture. Three-year-old callus tissues were used as samples. Callus tissue samples grown on 4.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and on 4.0 and 8.0 mg/LN-(2-furanylmethyl)-1H-purine-6 amine (kinetin) were cut and transferred to fresh medium containing 4.0 mg/L 2,4-D and 4.0, 8.0, 16.0, and 32 mg/L kinetin combinations. The pattern of changes observed in the ADH and mMDH isozymes as well as the growth of callus tissues was independent of the concentrations tested. The various ADH and mMDH isozymes seem to be products of differential association of subunits of the twoAdh and twomMdh genes. Both genes are active throughout callus tissue development; however, gene expression changed with various callus culture conditions. This study addresses how long-term callus culture conditions affect constitutive and differential gene expression of theAdh andmMdh genes inC. peruvianus.     

Valepotriates accumulation in callus, suspended cells and untransformed root cultures of Valeriana glechomifolia

Summary Valeriana glechomifolia is an endemic species of southern Brazil, capable of accumulating, in all of its organs, the terpene derivatives known as valepotriates, the presumed sedative components of the roots of pharmaceutically used species of Valeriana. In vitro cultures of the plant were established and the accumulation of acevaltrate, didrovaltrate, and valtrate in callus, cell suspension, and untransformed root cultures was studied. Leaves of in natura plants and roots of micropropagated plantlets were used as the explants for callus induction and root culture establishment, respectively, on Gamborg B5 basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or with kinetin (KIN). Culture growth and secondary metabolite yields were enhanced with 2,4-D (4.52μM) and KIN (0.93μM). Maximum valepotriate contents, quantified by HPLC, of acevaltrate (ACE) 2.6mg g⁻¹ DW, valtrate (VAL) 10.2mgg⁻¹ DW, and didrovaltrate (DID) 2.9mg g⁻¹ DW were observed in root cultures after 7–8wk of culture.