LIGHT AND HORMONAL CONTROL OF ROOT FORMATION IN ZEA MAYS CALLUS CULTURES

Callus cultures of Zea mays were used to study the interaction of light with exogenous cytokinin/auxin levels in the initiation, growth and development of roots. Three auxins, indoleacetic acid (IAA), naphthaleneacetic acid (NAA) and 2,4 dichlorophenoxyacetic acid (2,4 D) were remarkably different in their effects on callus growth and root initiation. NAA at concentrations of 5 and 25 μM produced the highest combined yields of callus and roots under low light conditions. No significant morphological effects on roots were observed with the three auxins tested nor did low and intermediate light intensities alter root development.
At intermediate light levels the addition of the cytokinin, zeatin, was also able to influence the differentiation of the callus tissue. Increasing the cytokinin/auxin ratio from low to high shifted the development from callus growth to abundant root formation. High light caused the formation of short, thick roots. This effect could be counteracted in part by zeatin which promoted elongation. These observations suggest that both, the cytokinin/auxin ratio and light play an important role in the development of monocotyledonous roots.     

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.     

The regeneration potential of wheat shoot meristems in the presence and absence of 2,4-dichlorophenoxyacetic acid

Summary Tissue cultures capable of plant regeneration were successfully initiated from extremely immature shoot meristems of 21 randomly selected genotypes of wheat on nutrient media containing 2,4-dichlorophenoxyacetic acid (2,4-D). By means of scanning electron microscopy it was demonstrated that cultures consisted of teratomatous primordia, which were kept in a proliferating budding state by the 2,4-D. These are characteristic of cereal tissue cultures. Release of the primordia and outgrowth of normal shoots and roots occurred when the cultures were no longer exposed to 2,4-D. Shoot primordia which were clearly identifiable were always associated with root primordia in a quasi-bipolar fashion. Sometimes regions assumed the shape of zygotic embryos, but the transition from apparently normal embryos with scutellum to abnormal configurations with shoot and root regions was gradual. The differences between genotypes in shoot regeneration potential was minimal compared to cultures derived from explants which were taken from regions temporally and spatially more distant from the shoot apex. It is concluded that the ability to give rise to cultures capable of shoot regeneration was lost within a fraction of a millimeter distance from the apical meristem in many genotypes. The proliferating tissues were subcultured at regular intervals over a period of one year and the regeneration potential was monitored. Areas capable of shoot regeneration tended to deteriorate more or less rapidly and were overgrown by root-type tissue in a number of genotypes. The results are discussed in the context of the frequently observed, but largely unexplained, variability in the regeneration potential of cereal tissue cultures.     

Induction and cultures of mountain ginseng adventitious roots and AFLP analysis for identifying mountain ginseng

Among the various plant growth regulators (PGR), 9.0 μM 2,4-D(2,4-dichlorophenoxyacetic acid) turned out to be the best for callogenesis and 4.5 μM 2,4-D was revealed to be more suitable for callus proliferation. Of the four basal media, the highest number of 8.2 adventitious roots was induced in Woody plant medium (WPM) containing 14.8 μM IBA(indole 3-butyic acid). F tests on each auxin treatment in the ANOVA revealed highly significant difference at α=0.01. The number of multiple adventitious roots (MARs) formed in each adventitious roots highly depended on the types and levels of auxin tested. At both 24.6 μM and 34.4 μM IBA, more than 25 MARs were induced after 4 weeks of culture. When different medium were applied, immersion type showed the highest fresh weight of MARs at the time of harvest. With 160 g fresh weight of inoculum, about 14.2 times of biomass was obtained after 6 weeks of culture. Identical fractionation pattern of the amplified fragment length polymorphism (AFLP) analysis was observed between the adventitious root and donor of a mountain ginseng. This observation suggested that any rearrangements of the genomic DNA had not occurred in the adventitious roots after 1 year of cultures. The cultivated ginseng and the mountain ginseng groups could be distinguished by 2 AFLP markers.     

Somatic embryogenesis in tulip (<Emphasis Type="Italic">Tulipa gesneriana</Emphasis> L.) flower stem cultures

In the present study, the procedures for induction of somatic embryogenesis (SE) in an in vitro culture of the tulip have been developed. SE was initiated on flower stem explants isolated from “Apeldoorn” bulbs during their low-temperature treatment. Bulbs had not been chilled or had been chilled for 12 or 24 weeks at 5°C. The explants were cultured with exogenous auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (Picloram), α-naphthaleneacetic acid (NAA) at 1–100 μM and cytokinins: benzyladenine (BA) and zeatin (ZEA) at 0.5–50 μM. Increase in auxin concentrations caused an intensive enlargement of the explant parenchyma, which changed into homogenous colorless callus. On the same media, vein bundles developed into yellowish, nodular callus. Picloram was more efficient in inducing the formation of embryogenic nodular callus than 2,4-D, whereas the latter stimulated formation of colorless callus. The base of the lower part of the flower stem isolated from bulbs chilled for 12 weeks proved to be the best explant for callus formation. The highest number of somatic embryos was produced on medium with 25 μM Picloram and 0.5 μM BA. Development of adventitious roots was noticed in the presence of 2,4-D. Globular embryos developed into torpedo stage embryos under the influence of BA (5 μM) and NAA (0.5 μM). Morphological and anatomical data describing development of callus and somatic embryos are presented.     

<Emphasis Type="Italic">In vitro</Emphasis> rooting of microshoots of <Emphasis Type="Italic">Cotinus coggygria</Emphasis> Mill,a woody ornamental plant

Studies on rooting of microshoots of smokebush (Cotinus coggygria Mill, var. Royal Purple), a woody ornamental, were carried out in vitro. Microshoots were rooted in a mixed-auxin regime (indole 3-acetic acid, indole butyric acid [IBA], and naphthalene acetic acid) or singly in the above auxins and the 2,4 dichlorophenoxyacetic acid (2,4-D) over a wide concentration range. Indole butyric acid at 10 μM proved to be the most suitable treatment, producing less basal callus, 100% rooting, and earlier root emergence than the other treatments. No roots were formed with 2,4-D. A 6-day root induction period was obtained with 10 μM of IBA. Histological studies revealed increased mitotic activity after 3 d in culture in the medullary ray cells, which led to root primordium formation, several of which were formed simultaneously around the base of the explant. The vascular tissues of these primordia connected to those of the explant, and roots began to emerge from the base by day 10. Thus, direct rhizogenesis occurred with the IBA treatment, as opposed to the roots that were formed in the basal callus under the mixed-auxin regime.     

Wheat Callus Culture: the Initiation, Growth and Organogenesis of Callus Derived from Various Explant Sources

Callus was obtained from mature excised embryos of wheat, fromnodal and internodal stem segments and from rachis segmentsusing the medium of Murashige and Skoog(1962)(M medium), containing1-0mg l^–1 2,4-D, and from immature embryos using the mediumof Green and Phillips (1975) containing 2 mg l^–1 2,4-D.Callus yield from mature embryos depended upon the cultivarused. No callus could be obtained from leaf segments. Callusderived from mature embryos and nodal stem segments was successfullymaintained by serial sub-culture on the M medium containing2,4-D for up to 3 years although its growth rate declined toa lower level as culture proceeded. Such cultures consistently produced roots when transferred toa medium containing a low level of 2,4-D or no 2,4-D. The presenceof the auxin was essential for continued proliferation of thecallus tissue. Shoot initiation was infrequent, did not occurafter the first few sub-cultures and could not be enhanced byvarious auxin and cytokinin additions to the medium. Callusderived from immature embryos did not have an enhanced potentialfor shoot initiation. Triticum aestivum, wheat, callus culture, organogenesis     

Effects of cytokinin on adventitious root formation in callus cultures ofVigna unguiculata (L.) walp

Summary Yellowish compact callus, induced from cowpea hypocotyls on Murashige and Skoog(MS) medium (1962) containing 0.2 mg/l(0.93 μM) kinetin and 0.4 mg/l (1.81 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), was subcultured on MS medium containing cytokinin alone, auxin alone, or auxins plus cytokinins in order to determine the effect of cytokinins on root organogenesis in callus cultures. The callus actively proliferated on the same medium but did not show any organogenic activity macroscopically as well as microscopically. On medium with N⁶-benzyladenine (BA) and 1-naphthaleneacetic acid (NAA), the yellowish compact callus first changed to pale green compact callus and then many green spots appeared on its surface under light culture. But the yellowsih compact callus remained yellowish and white spots appeared on its surface in dark culture. These spots gradually became white nodular structures. Adventitious root formation from the nodular structures occurred not only on the same medium, but also on medium with either auxin or cytokinin but not both. Yellowish compact callus on medium with auxin alone was transformed to yellowish friable callus, which did not develop adventitious roots. The yellowish friable callus could gain rhizogenic activity only after morphological modification to pale green compact callus on medium with auxin plus cytokinin. The modified callus did not form adventitious roots on medium with auxins but only with cytokinins. Therefore, it is suggested that cytokinins have stimulating effects on root formation from callus that previously did not show rhizogenic activity on medium with auxins alone. In addition, the rhizogenic potential of cowpea callus was discriminated from that of leaf explants, which formed adventitious roots directly on medium with auxin alone.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration through somatic embryogenesis and organogenesis using cotyledons of <Emphasis Type="Italic">Cassia angustifolia</Emphasis> Vahl

In vitro regeneration through somatic embryogenesis as well as organogenesis using cotyledon of a woody medicinal legume, Cassia angustifolia is reported. The cotyledons dissected from semi-mature seeds, if inoculated on Murashige and Skoog’s medium (MS) supplemented with auxin alone or in combination with cytokinin, produced direct and indirect somatic embryos. A maximum of 14.36 ± 2.26 somatic embryos per 20 mg of explants including callus were produced in 70% cultures on MS medium with 2.5 μM benzyladenine (BA) + 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Although the percentage of embryogenic cultures was higher (83.33%) at 10 μM 2,4-D + 1 μM BA, the average number of somatic embryos was much less (7.6 ± 0.85) at this level, whereas at 2.5 μM BA and 5 μM 2,4-D, there was a simultaneous formation of both somatic embryos and shoots. The somatic embryos, although started germinating on the same medium, developed into full plantlets only if transferred to MS basal with 2% sucrose. Cytokinins alone did not induce somatic embryogenesis, but formed multiple shoots. Five micromolar BA proved optimum for recurrently inducing shoots in the competent callus with a maximum average of 12.04 ± 2.10 shoots and shoot length of 2.26 ± 0.03 cm. Nearly 91.6% shoots (2–2.5 cm in size) organized an average of 5.12 ± 0.58 roots on half strength MS + 10 μM indole-3-butyric acid. All the plantlets have been transferred successfully to soil. Types of auxin and its interaction with cytokinin significantly influenced somatic embryogenesis.     

Induction of embryogenicTriticum aestivum L. calli. II. Quantification of organic addenda and other culture variable effects

Nine experiments were conducted to determine effects of various culture medium addenda on induction of embryogenic calli from immature embryos of a responsiveTriticum aestivum L. genotype (PCYT 10). Effects were quatified by counting somatic embryos (embryoids) per callus. Optimal auxin concentrations to induce and maintain somatic embryogenesis were 3.62 M 2,4-dichlorophenoxyacetic acid (2,4-D) or 9.05 M 3,6-dichloro-o-anisic acid (dicamba). In general, dicamba permitted formation of significantly more embryoids than 2,4-D. Kinetin (6-furfurylaminopurine) at 2.56 M or 4.65 M significantly increased percentage scutellar callus when added to 2,4-D or dicamba-containing medium, respectively. Kinetin at 4.65 M signficantly increased the numbers of embryoids formed when added to medium containing either synthetic auxin. Significantly fewer embryoids formed when cultures were incubated under diffuse light (16-h photoperiod). Casein hydrolysate (200 mgl^-1) or L-arginine (0.23 mM) had no effect on numbers of embryoids formed, whereas L-tryptophan (0.20 mM) enhanced such formation with 2,4-D and decreased such formation with dicamba. Two additional experiments generally demonstrated that response to auxin source in the genotypes ND 7532, PCYT 20, Yaqui 50, and Oasis was similar to that in PCYT 10. The higher molar concentration of dicamba required to induce embryogenic callus coupled with more evident embryoid precocious germination and a more rapid rate of tissue necrosis upon extended incubation without subculture suggests that dicamba is metabolized more rapidly than 2,4-D inT. aestivum callus cultures.This study was supported by NASA-Ames Cooperative Agreement No. NCC2-139. Contribution of the Utah Agricultural Experiment Station, Utah State University, Logan, UT, Journal Paper No. 3359.     

Reciprocal Antagonism between the Herbicides, Diclofop-Methyl and 2,4-D, in Corn and Soybean Tissue Culture

The antagonistic interaction between the grass herbicide, diclofopmethyl (methyl 2-[4(2′,4′-dichlorophenoxy)phenoxy]propanoate) (DM), and 2,4-dichlorophenoxyacetic acid (2,4-D), was demonstrated in DM-resistant soybean (Glycine max [L.] Merr.) and DM-susceptible corn (Zea mays L.). 2,4-D caused root shortening and thickening, and induced callus growth in soybean and corn root tissue cultures at 1 and 10 micromolar. Normal soybean root growth was unaffected by 10 micromolar DM whereas corn root growth was inhibited completely by 1 to 10 micromolar DM. DM at 10 micromolar reversed completely the induction of callus growth by 1 micromolar 2,4-D in soybean roots. In corn, 10 micromolar 2,4-D reversed the growth inhibiting activity of 1 micromolar DM and induced callus growth. The antagonistic interaction between DM and 2,4-D was reciprocal and the activity of either compound depended upon the relative concentration of the other. 2,4-D did not antagonize or decrease the activity of DM by decreasing its uptake by root tissues or increasing the rate of its detoxication. The antagonistic interaction between DM and 2,4-D probably involves involves cellular activity associated with actively growing and proliferating cells and requires the presence of both compounds at the sensitive site.     

Induction of embryogenic Triticum aestivum L. calli. II. Quantification of organic addenda and other culture variable effects

Nine experiments were conducted to determine effects of various culture medium addenda on inducation of embryogenic calli from immature embryos of a responsive Triticum aestivum L. genotype (PCYT 10). Effects were quantified by counting somatic embryos (embryoids) per callus. Optimal auxin concentrations to induce and maintain somatic embryogenesis were 3.62 M 2,4-dichlorophenoxyacetic acid (2,4-D) or 9.05 M 3,6-dichloro-o-anisic acid (dicamba). In general, dicamba permitted formation of significantly more embryoids than 2,4-D. Kinetin (6-furfurylaminopurine) at 2.56 M or 4.65 M significantly increased percentage scutellar callus when added to 2,4-D or dicamba-containing medium, respectively. Kinetin at 4.65 M significantly increased the numbers of embryoids formed when added to medium containing either synthetic auxin. Significantly fewer embryoids formed when cultures were incubated under diffuse light (16-h photoperiod). Casein hydrolysate (200 mg1^-1) or L-arginine (0.23 mM) had no effect on numbers of embryoids formed, whereas L-tryptophan (0.20 mM) enhanced such formation with 2,4-D and decreased such formation with dicamba. Two additional experiments generally demonstrated that response to auxin source in the genotypes ND 7532, PCYT 20, Yaqui 50, and Oasis was similar to that in PCYT 10. The higher molar concentration of dicamba required to induce embryogenic callus coupled with more evident embryoid precocious germination and a more rapid rate of tissue necrosis upon extended incubation without subculture suggests that dicamba is metabolized more rapidly than 2,4-D in T. aestivum callus cultures.This study was supported by NASA-Ames Cooperative Agreement No. NCC2-139. Contribution of the Utah Agricultual Experiment Station, Utah State University, Logan, UT, Journal Paper No. 3358.     

Auxin effects on rooting in pea cuttings

Light-grown stem cuttingss of Pisum sativum L. cv. Weibull's Marma were rooted in a nutrient solution. The presence of 10 μ M indolylacetic acid (IAA) in the solution for 24 h or longer periods decreased the number of roots subsequently formed to about 50% of control, provided IAA was present in the solution during any of the 4 first 24 h periods. Treatment for 6 h or shorter periods caused no or small response. IAA did not appreciably change the time needed for root formation, the time course of root appearance or the pattern of root distribution along the basal internode. IAA at 100 μ M usually increased the number of roots although variable results were obtained with this IAA concentration.
The number of roots was strongly increased by treatment with indolylbutyric acid (IBA) or 2,4-dichlorophenoxyacetic acid (2,4-D). None of these or other synthetic auxins decreased the number of roots in suboptimal concentrations. Experiments with 10 μ M IBA showed that stimulation of rooting was obtained only if the auxin was present in the rooting solution for several days. Simultaneous treatment with IAA decreased the stimulating effect of IBA to some extent, whereas no such response was obtained if IAA was combined with 2,4-D.
IAA applied in lanolin to the stem of intact cuttings decreased the number of roots formed. Decapitation and debudding of the cuttings decreased the number of roots formed. If at least 2 leaves were left this decrease was efficiently counteracted by an optimal IAA dose applied to the upper part of the stem. A five times higher dose was less effective, indicating a negative effect on rooting also by IAA applied to the shoots.     

Genetic analysis of adventitious root formation with a novel series of temperature-sensitive mutants of Arabidopsis thaliana

When cultured on media containing the plant growth regulator auxin, hypocotyl explants of Arabidopsis thaliana generate adventitious roots. As a first step to investigate the genetic basis of adventitious organogenesis in plants, we isolated nine temperature-sensitive mutants defective in various stages in the formation of adventitious roots: five root initiation defective (rid1 to rid5) mutants failed to initiate the formation of root primordia; in one root primordium defective (rpd1) mutant, the development of root primordia was arrested; three root growth defective (rgd1, rgd2, and rgd3) mutants were defective in root growth after the establishment of the root apical meristem. The temperature sensitivity of callus formation and lateral root formation revealed further distinctions between the isolated mutants. The rid1 mutant was specifically defective in the reinitiation of cell proliferation from hypocotyl explants, while the rid2 mutant was also defective in the reinitiation of cell proliferation from root explants. These two mutants also exhibited abnormalities in the formation of the root apical meristem when lateral roots were induced at the restrictive temperature. The rgd1 and rgd2 mutants were deficient in root and callus growth, whereas the rgd3 mutation specifically affected root growth. The rid5 mutant required higher auxin concentrations for rooting at the restrictive temperature, implying a deficiency in auxin signaling. The rid5 phenotype was found to result from a mutation in the MOR1/GEM1 gene encoding a microtubule-associated protein. These findings about the rid5 mutant suggest a possible function of the microtubule system in auxin response.     

The effects of diclofop-methyl on root growth of wild oat

Diclofop-methyl severely reduced the growth of seminal roots of wild oat ( Avena fatua L.) when applied in hydroponics at 0.01 and 0.05 μ M . Lateral roots emerged closer to the seminal root apex than in the controls, but coronal root number and length were unaffected at 0.01 μ M . However, doses of 0.05 to 0.1 μ M induced more but shorter coronal roots to emerge than for controls. At 1 μ M the number and length of coronal roots were less than for controls. Root-applied diclofop-methyl at 1 μ M inhibited emerging second leaf growth to the same extent as a foliar dip in 1 μ M diclofop-methyl without causing chlorosis as foliar treatment does. Because of limited basipetal transport of foliarly-applied diclofop-methyl, shoot treatment was ineffective in inducing abnormal root morphogenesis of the seminal and lateral root systems, although it caused abnormalities of the coronal root system. Time course studies were initiated to examine the effect of root-applied diclofop-methyl at 0.05 μ M . Seminal root growth was inhibited (by diclofop-methyl) soon after treatment, while controls continued elongating. The distance between the seminal root apex and the first lateral primordia increased in the controls within one day after treatment, but decreased in the herbicide-treated roots. The distance between the seminal root apex and the first emerged lateral root was reduced by three days after treatment. The number of lateral primordia and emerged roots was unaffected three days after treatment. These dose-response and kinetic results suggested that diclofop-methyl caused a loss of apical dominance in the seminal root.     

Tissue Culture of Maize I. Callus Induction and Growth

Callus induction and subculture was successful with mature embryos and stem sections of seedlings of Zea mays L. on Linsmaier and Skoog's medium modified to contain 4 mg/I of 2,4-D and 1 g/I of casamino acids. — 2,4-D was superior to NAA and IAA for both callus induction and growth. Callus subcultured on NAA formed abundant roots on agar-solidified media and numerous root-like primordia in liquid cultures. — Kinetin had no effect on callus induction in the presence of 2,4-D and neither kinetin nor gibberellic acid stimulated callus growth during subculture. — Callus grew equally well on the medium of Linsmaier and Skoog, that of Schenk and Hildebrandt, and the B-5 medium of Gamborg and Eveleigh containing 2% sucrose, 4 mg/I of 2,4-D and 1 g/I of casamino acids. — The callus grew more rapidly at 25°C than at 30°C or 35°C. Little difference was noted at any temperature in callus growth in alternating light (16 h) and dark (8 h) or continuous dark. — Sucrose was superior to glucose and maltose in both liquid and agar-solidified cultures. Lactose and galactose failed to support callus growth.     

Enhanced shoot organogenesis in Cassia angustifolia Vahl. — a difficult-to-root drought resistant medicinal shrub

In vitro regeneration was achieved through callus culture derived from cotyledon explants of Cassia angustifolia Vahl. on MS (Murashige and Skoog, 1962) medium. Calli were induced from cotyledon explants excised from aseptic 14?days old seedlings on MS medium containing 2,4-D (2,4-dichlorophenoxy acetic acid) and 2,4,5-T (2,4,5-trichlorophenoxy acetic acid) at different concentrations with 3% sucrose and 0.8% agar. Optimal growth of callus was obtained at 5.0???M 2,4-D, which was proved to be the best for shoot regeneration when sub cultured onto MS medium supplemented with cytokinins either alone or in combination with an auxin. Maximum number of shoots (23.2?±?1.4) were produced at 5.0???M 6-benzylaminopurine (BA) and 0.4???M ??-naphthalene acetic acid (NAA). Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 1.0???M indole-3-butyric acid (IBA) and 5.0???M phloroglucinol (PG). Rooted plantlets thus developed were hardened and successfully established in the soil. This protocol yielded an average of 23 plants per cotyledon explant over a period of 4?months.     

The effect of plant growth regulators on growth, morphology and condensed tannin accumulation in transformed root cultures of Lotus corniculatus

This study concerns the effects of four different classes of plant growth regulators on root morphology, patterns of growth and condensed tannin accumulation in transgenic root cultures of Lotus corniculatus L. (Bird's-foot trefoil). Growth of transformed roots in 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in decreased tannin levels relative to controls at concentrations of 10^-6 M and above, while gibberellic acid (GA₃) inhibited tannin accumulation at concentrations of 10^-7 M and above. Benzyladenine (BA) had little effect at low concentrations (10^-7 M and below) but resulted in an increase in tannin levels at 10^-6 M. Abscisic acid had little effect on levels of condensed tannins at any of the concentrations used. Experiments involving growth regulator addition and medium transfer demonstrated that 2,4-D inhibition of tannin accumulation could be reversed by GA₃ and BA, while GA₃ downregulation could only be reversed by the addition of 2,4-D. Although 2,4-D inhibited tannin accumulation, addition of 2,4-D to root cultures grown for 14 or 28 days in the absence of plant growth regulators stimulated both growth and tannin biosynthesis. Characteristic alterations in root morphologies accompanied growth regulator-mediated modulation of tannin biosynthesis. Growth in 2,4-D resulted in partially de-differentiated root cultures while growth in GA₃ produced roots with an elongated phenotype. Restoration of tannin biosynthesis in 2,4-D-treated roots was accompanied by root re-differentiation and the production of new lateral roots.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid 3 - FW fresh weight     

Genotypic Responses to Auxins in Tissue Cultures of Phaseolus

Responses of seven Phaseolus genotypes to the four auxins picloram, 2,4-D, NAA and 1AA, in tissue cultures, were examined. Callus growth was promoted by picloram and the range of effective concentrations for most genotypes was broad. The auxin 2,4-D also enhanced callus growth, but the range of optimal concentrations was markedly narrower than that of picloram. NAA when supplied at relatively high concentrations gave good growth. IAA was ineffective in supporting callus growth. The differences in 2,4-D concentrations required for optimal growth and the differential responses to low concentrations (0.04–1.25 μM) of picloram between several genotypes tested were large. These genotypic variations in auxin responses were repeatable and may thus reflect genetic differences.     

Lignin release and photomixotrophism in suspension cultures of Picea abies

The effect of different concentrations of sucrose (0-4%) and of two growth regulators (0–50 μ M 2,4-D and 0–25 μ M kinetin) was tested on growth and chlorophyll content of suspension cultures of Picea abies (L.) Karst. originating from chlorophyllous embryo callus in an elevated CO₂ (2%) atmosphere. A continuous chlorophyllous suspension culture was achieved on a medium containing 2% sucrose and a low level of organic nitrogen (0.25 m M arginine and 0.5 m M glutamine) supplemented with 2,4-D (0.5 μ M ) and kinetin (2.5 μ M ). The same medium with 4% sucrose gave the best growth response, but a negative correlation between chlorophyll level and growth was observed. The chlorophyllous cultures grew slowly in a medium with low (0.5%) sucrose or without any carbohydrate source, suggesting photomixotrophism. A high concentration of kinetin inhibited both growth and chlorophyll synthesis. Release of lignin into the nutrient medium was observed in several experiments, especially in slow-growing cultures supplemented with sucrose. Only a few successive passages of suspensions that produced lignin could be cultured before cell death. The cultures releasing lignin may be unique for studies on synthesis and biodegradation of this very complex compound.