Hildebrandt Medium Research Articles

Phenolics from Cell Suspension Cultures of Penstemon serrulatus; Relation to Plant Organs

Abstract By repeated selection of pigment portions of tissue the red callus induced from root seed­lings of Penstemon serrulatus Menz. was chosen for suspension culture, which was maintained in Schenk and Hildebrandt medium supplemented with naphthaleneacetic acid (0.2 mg/l), 6-benzylaminopurine (2 mg/l) and sucrose (50 g/l). From the cultured cells eight phenolic compounds were isolated. They were identified as cyanidin 3-O-glucoside, delphinidin 3-O-glucoside, luteolin, luteolin 7-O-glucoside, norartocarpetin 7-O-glucoside, verbascoside, martynoside and leucosceptoside A. The kind of cell line, its age and light irradiation were important factors in flavonoid production, but production of phenylpropanoid glycosides was found to be unaffected by these factors. The phenolic composition found in the cell culture was compared with those in the flowers and leaves of original plants of P. serrulatus.

Embryogenic Callus Induction in Fraser Fir

Mass propagation of Fraser fir [Abies fraseri (Pursh) Poir], a valuable Christmas tree species in the United States, is problematic because methods currently used are inadequate. According to our results with somatic embryogenesis, the culturing methods used with other Abies species are applicable to Fraser fir. Stage 1 somatic embryogenic callus, characterized by suspensor cells and embryo heads, was obtained at low frequency using Schenk and Hildebrandt medium supplemented with 5 mm glutamine, 0.05% casein hydrolysate, 0.01% myoinositol, 2% sucrose, 5 μM benzyladenine, and 0.6% agar. The developmental stage of the embryo was important; embryogenic callus was obtained only with immature, precotyledonary embryos, not with fully formed embryos. Cold storage of cones containing immature embryos inhibited callus proliferation. Genotype was significant in that 35 of 44 families tested proliferated callus; however, only one embryo within one family continued proliferation to produce stage 1 embryogenic callus. Fully formed somatic embryos were not produced because the callus did not continue to proliferate. Although these experiments met with only limited success, they demonstrate the potential for somatic embryogenesis in Fraser fir and the general applicability of methods used with other Abies species.

Micropropagation of Sego Lily

The Sego lily (Calochortus nuttallii T. & G.) produces showy flowers and is a wild species indigenous to the western United States. A 3- to 5-fold increase in shoots per month was achieved when basal sections of bulbs were cultured on Schenk and Hildebrandt medium containing 88 mM sucrose and 8.9 µM 6-benzyladenine (BA) and subcultured every 28 d on the same medium but containing 88 mM sucrose, 2.2 µM BA, and 1.0 µM indole-3-butyric acid. Roots formed from 94% of shoots cultured at 13 °C on medium containing 88 mM sucrose and 2.7 µM α-naphthalene acetic acid. Bulbs formed at the base of shoots cultured on medium containing 263 mM sucrose.

Plant regeneration from juvenile and adult Anthyllis cytisoides, a multipurpose leguminous shrub

Anthyllis cytisoides , a legume shrub used for afforestation and reclamation of degraded Mediterranean areas, was successfully micropropagated from expiants of juvenile (cotyledonary nodes and apical buds) and adult origin (axillary buds). Multiple shoot formation was dependent on the presence of benzyladenine in the induction medium. Of the salt formulation and expiants examined, the higher proliferation rates were obtained when axillary buds from adult plants were cultured on a modified Schenk and Hildebrandt medium. Following the preferred protocol, shoot yield reached values greater than 100 shoots per expiant. Plants were easily rooted and transplanted into greenhouse.

Enhancement by Osmotic Treatment of Hairy Root Transformation of Alfalfa Suspension Cultures, and Chromosomal Variation in the Transformed Tissues

Seeds of alfalfa were germinated on Murashige and Skoog medium without phytohormones. The hypocotyls ofseedlings were excised and cultured on the same medium with 2,4- dichlorophenoxyacetic acid (9.05 mM) to induce callus. Granular calluses were suspended and cultured in Schenk and Hildebrandt medium supplemented with 2.26 mM 2,4-dichlorophenoxyacetic acid on a shaker at 80 rpm. Alfalfa suspension cultures were treated with 0.45 M mannitol for 1 h, and washed twice with 0.16 M CaCl2 .2H2 O. After pretreatment, the pellets were resuspended in Schenk and Hildebrandt medium without phytohormone (10 mL g-1 suspension cultures), and 0.2 mL of Agrobacterium rhizogenes suspension was added. A mixture of alfalfa cells and Agrobacterium was co-cultured at 25 ± 2˚C for 2 days. Transformants (transformed calluses and hairy roots) were obtained by hormone-free selection. Several factors, such as culturing stage of suspension cultures, phytohormone constitution of suspension medium and basal selection medium of transformants, affected the transformation frequency remarkably. Paper electrophoresis revealed that over 70% of transformants could synthesise agropine and mannopine. A comparative cytological analysis revealed the number and structural alterations of chromosomes in the resulting materials. The transformed tissue was not able to be regenerated, possibly due to chromosomal abnormalities.

Effects of medium components and light on callus induction, growth, and frond regeneration in Lemna gibba (Duckweed)

Basal media, plant growth regulator type and concentration, sucrose, and light were examined for their effects on duckweed (Lemna gibba) frond proliferation, callus induction and growth, and frond regeneration. Murashige and Skoog medium proved best for callus induction and growth, while Schenk and Hildebrandt medium proved best for frond proliferation. The ability of auxin to induce callus was associated with the relative strength of the four auxins tested, with 20 or 50 µM 2,4-dichlorophenoxyacetic acid giving the highest frequency (10%) of fronds producing callus. Auxin combinations did not improve callus induction frequency. Auxin in combination with other plant growth regulators was needed for long-term callus growth; the two superior plant growth regulator combinations were 10 µM naphthaleneacetic acid, 10 µM gibberellic acid, and 2 µM benzyladenine with either 1 or 20 µM 2,4-dichlorophenoxyacetic acid. Three percent sucrose was best for callus induction and growth. Callus induction and growth required light. Callus that proliferated from each frond’s meristematic zone contained a mixture of dedifferentiated and somewhat organized cell masses. Continual callus selection was required to produce mostly dedifferentiated, slow-growing callus cell lines. Frond regeneration occurred on Schenk and Hildebrandt medium without plant growth regulators but was promoted by 1 µM benzyladenine. Callus maintained its ability to regenerate fronds for at least 10 mo. Regenerated fronds showed a slower growth rate than normal fronds and a low percentage of abnormal morphologies that reverted to normal after one or two subcultures.

Wide hybridization between Brazilian soybean cultivars and wild perennial relatives

Employing a different culture strategy, we obtained a greatly improved frequency of embryo rescue in intersubgeneric soybean hybrids. Successful crosses were obtained in 31 different genotype combinations between nine Brazilian soybean lines as the female parents and 12 accessions from Glycine canescens, G. microphylla, G. tabacina and G. tomentella. The hybrid pod retention rate dropped to about 10% during the first 8 days after pollination and stayed largely unchanged up to the 20th day. Immature harvested seeds fell into three size groups: Group 1, smaller than 1.3 mm (mostly empty seed coats); Group 2, 1.9-5.0 mm; Group 3, larger than 5 mm (from selfing). A total of 90 putative hybrid embryos were rescued using a highly enriched B5 medium to nourish the newly dissected embryos. The growing embryos were then placed in a high osmotic, modified B5 medium to induce maturation and dormancy. Schenk and Hildebrandt medium was used to germinate the dormant, partially dehydrated, physiologically mature embryos. Approximately 37% of the rescued embryos developed into plantlets in vitro, and approximately 8% grew into mature plants in the greenhouse. Morphological, cytological and isoenzyme patterns confirmed the hybrid status of all seven mature plants, all of which were generated using G. tomentella G 9943 as the paternal parent. It was observed that all soybean lines crossed with G 9943 were capable of producing mature hybrid plants. There was no correlation between the initial size of Group 2 seeds and plant survival rate. The hybrids were cloned by grafting and treated with colchicine. One of the treated plants displayed chromosome doubling.

Somatic embryogenesis and plant regeneration in Floribunda rose ( Rosa hybrida L.) cvs. Trumpeter and Glad Tidings

Somatic embryogenic callus was initiated from in vitro-derived petiole and root explants, but not leaves, of the Floribunda rose cultivars Trumpeter and Glad Tidings following repeated subculture of callus on Schenk and Hildebrandt (SH) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). The use of a high auxin pretreatment increased the frequency of somatic embryogenesis, whilst l-proline, as a media supplement, had a critical role in enhancing somatic embryogenesis in the early stages of culture but increased the frequency of abnormal embryo formation when used in the later stages of culture. Maturation of somatic embryos was achieved by transfer to SH medium with reduced concentrations of 2,4-D and incorporation of abscisic acid (ABA) and 6-benzylaminopurine (BAP). The use of SH medium containing BAP and indole-3-butyric acid (IBA) facilitated embryo germination which was also enhanced by cold treatment. Phenotypically normal plants were recovered from germinated somatic embryos and successfully transferred to the glasshouse for flowering.

Morphogenesis in leaf and single-cell cultures of mature Juniperus oxycedrus.

Single cells were mechanically isolated from leaf-derived callus of mature Juniperus oxycedrus L. These cells divided and gave rise to callus when plated on medium containing growth regulators. Best plating efficiency was obtained on a modified Schenk and Hildebrandt medium supplemented with 0.6 micro M 2,4-dichlorophenoxyacetic acid and 100 mg l(-1) casein hydrolyzate. Although single-cell-derived callus showed poor morphogenic potential, both adventitious shoots and embryogenic tissues differentiated from the callus. We also achieved induction of somatic embryogenesis in leaf explants of mature J. oxycedrus trees cultured in the presence of 6.0 or 10.0 micro M 2,4-dichlorophenoxyacetic acid or picloram. Frequency of embryogenic callus ranged from 6 to 18%; however, under the culture conditions tested, isolated embryos failed to develop into plants.

Elongation, rooting and acclimatization of micropropagated shoots from mature material of hybrid larch

Factors were defined for elongation, rooting and acclimatization of micropropagated shoots ofLarix x eurolepis Henry initiated from short shoot buds of plagiotropic stecklings serially propagated for 9 years from an 8-year-old tree. Initiation and multiplication were on Schenk and Hildebrandt (SH) medium supplemented with 5 μM 6-benzyladenine (BA) and 1 μM indole-butyric acid (IBA). Stem elongation was obtained in 36% of the shoots on SH medium containing 0.5 μM BA and 63% of the remaining non-elongated shoots initiated stem elongation after transfer on SH medium devoid of growth regulators. Rooting involved 2 steps: root induction on Campbell and Durzan mineral salts and Murashige and Skoog organic elements, both half-strength (CD-MS/2), supplemented with 1 μM of both naphthaleneacetic acid (NAA) and IBA, and root elongation following transfer to CD-MS/2 medium devoid of growth regulators. Repeating this 2-step sequence yielded up to 67% rooted shoots. Acclimatization of plantlets ranged from 83% to 100%. Over 300 plants were transferred to the greenhouse; some showed plagiotropic growth.

In vitro control of adventitious bud differentiation by inorganic media components in leaves of matureJuniperus oxycedrus

The effects of changes in the concentration of macronutrients on BA-induced caulogenesis from leaves of matureJuniperus oxycedrus cultured on modified Murashige and Skoog or Schenk and Hildebrandt media are reported. The bud-forming capacity of the explants depended mainly on the ratios among the levels of ammonium, nitrate, and potassium. The most favorable media formulations for differentiation of adventitious buds were those with nitrate:potassium, ammonium:potassium, and nitrate:ammonium ratios near 1, around 0.1, and between 9 and 15, respectively. The total ionic strength of the media limited bud induction, but only when a disequilibrium of these ratios was produced.

Axillary shoot proliferation in cultures of explants from mature Juniperus oxycedrus trees

We developed procedures for the micropropagation of Juniperus oxycedrus L. using shoot apices or nodal segments from mature plants. Of the media and explants examined, best culture establishment was obtained with shoot apices cultured on modified Schenk and Hildebrandt medium (SH medium) without growth regulators; however, shoot multiplication was only achieved when shoot apices isolated from shoots grown on SH medium without growth regulators were subcultured on SH medium containing 0.5 micro M benzyladenine. None of the auxins and methods tested for root induction provided satisfactory results.

Growth of axenic cultures of Cronartium flaccidum on callus tissue from Pinus nigra var. laricio and Pinus sylvestris

SummaryThe callus‐fungal method was employed to test the response to C. flaccidum of the highly susceptible P. nigra var. laricio and the resistant P. sylvestris, and to ascertain whether results obtained with this method matched in planta observations. Calli were inoculated with axenie cultures of C. flaccidum obtained by incubating basidiospores on modified Schenk and Hildebrandt medium. Several parameters were evaluated. Colony growth was more rapid on P. nigra var. laricio. Colonies were dense on P. nigra var. laricio, but sparse on P. sylvestris. Aerial hyphae growth was abundant on P. nigra var. laricio, but less frequent on P. sylvestris. Hyphal branching began after 18 h on P. nigra var. laricio and after 45 h on P. sylvestris. Necrosis of the host cells set in after 24 h on P. nigra var. laricio, and after 70 h on P. sylvestris. The number of cells with plasmolysis was much larger in P. nigra var. laricio than in P. sylvestris. These results were consistent with the known resistance of the two species on whole plants.

Effect of various bud induction treatments on elongation and rooting of adventitious shoots of Canary Island pine (Pinus canariensis)

Three-day-old cotyledonary explants of Pinus canariensis were subjected to 30 induction treatments using half-strength Bornman's medium containing various combinations of N6- benzyladenine, zeatin, kinetin and 2-isopentenyl-adenine. The highest numbers of buds were obtained with 10 μM 6-benzyladenine, but both kinetin and zeatin influenced shoot elongation. Shoots were maintained on half-strength Schenk and Hildebrandt medium with 2% sucrose and 0.05% activated charcoal. For rooting, shoots were pulsed for 4 h in a 100 μM indole-3-butyric acid aqueous solution (pH 4.2–4.5), and planted in peat:vermiculite:perlite (1:1:1). After 8 weeks, the numbers of rooted shoots were similar for most treatments. Therefore, the bud induction treatments did not significantly influence rooting of adventitious shoots of Canary Island pine.

Rapid micropropagation of Woodfordia fruticosa (L.) Kurz (Lythraceae), a rare medicinal plant

A rapid propagation method comprising initiation of in vitro shoot tip culture from field-grown flowering plants and reculture of the nodal segments of regenerated shoots in Schenk and Hildebrandt (1972) medium was developed for Woodfordia fruticosa (L.) Kurz., a rare medicinal shrub. A medium supplement of 6-benzylaminopurine (0.2 mg.l(-1)) induced high frequency (88%) development of axillary shoot buds (3.2) in 4-5 weeks. Subculture of the explants with multiple new shoots in fresh medium for 30 days yielded an even larger number (9.7) of shoots. Highest multiplication (26-35 shoots) was recorded when using culture initiation media with 0.5 mg.l(-1) each of BAP and NAA followed by subculture in 0.2 mg.l(-1) BAP. The shoot multiplication rate was further accelerated by reculturing 0.4-0.6 cm nodal segments of regenerated shoots in media with 1.0 mg.l(-1) BAP. Shoot cuttings (3.5-7.0 cm) were rooted in 0.2 mg.l(-1) IAA. Regenerated plants displayed uniform morphological, growth and flowering characteristics.

Somatic Embryogenesis and Plant Regeneration from Callus Cultures of ‘Tifton 9’ Bahiagrass

Identifying appropriate tissue culture and plant regeneration systems for agronomically important cultivars is often an important step in developing gene introduction techniques. Culture conditions for somatic embryogenesis and plant regeneration of ‘Tifton 9’ bahiagrass (Paspalum notatum Fluegge L.) from leaf‐stem cross sections are described. Cultures were initiated and maintained in the dark on Schenk and Hildebrandt medium containing 3% sucrose and 6.6 mg L−1 dicamba. Plant regeneration was initiated by transfer of callus tissue to medium lacking dicamba and incubating in continuous light. Scanning electron microscopy demonstrated, somatic embryogenesis as the process leading to plantlet formation. More than 96% of 4‐mm‐diam. callus segments produced plants, many with multiple germinating embryos. Approximately 300 plantlets were produced per gram of calli. Plants could be grown to maturity by transferring to greenhouse flats 3 wk after placing on regeneration medium. Nonspecific subculturing of primary calli resulted in a decline in regenerability and an increase in the number of albino regenerated plants after 4 to 5 mo from the initiation of callus growth on the explants. However, subculturing of specific callus cells identified as embryogenic extended the efficiency of regeneration and reduced the frequency of albino regenerates. Efficiency of regenerating green plants remained essentially unchanged for 10 mo. We have therefore identified a tissue culture and plant regeneration system suitable for use in genetic engineering.

In vitro micropropagation of Afghan pine

Clonal propagation by tissue culture of Afghan pine (Pinuseldarica Medw.) could be useful for improving Christmas tree production and afforestation of semiarid lands in North America. The objectives were (i) to develop organogenesis procedures for clonal propagation and (ii) to evaluate plantlet performance in the field. Shoot production from cotyledon explants was accomplished on solid Shenk and Hildebrandt medium amended with kinetin (11 μM) and benzyladenine (11 μM). Over 100 shoots per clone were obtained with this method. Spontaneous rooting was prevalent in some of the induced shoots. Shoots without root primordia were induced to form roots with naphthalene acetic acid (5 μM), indole butyric acid (10 μM), and benzyladenine (0.22 μM) in 1/2 strength Shenk and Hildebrandt medium. The rate of plantlet elongation in the field was similar to that of seedlings; however, survival of plantlets was lower than that of seedlings. The lower survival of plantlets was directly related to their smaller size at the time of planting. Organogenesis appears to be a very suitable method for mass micropropagation of this environmentally and economically useful tree.

Micropropagation of western hemlock (Tsuga heterophylla [Raf.] Sarg.) from embryonic explants

An in vitro propagation protocol is described for western hemlock, an important forestry species in Canada. For shoot bud induction, embryonic explants were placed initially on one-third strength Schenk and Hildebrandt medium containing 5 μM N6-benzyladenine (BA) or 5 μM BA in combination with either 5 μM kinetin or 5 μM 2-isopentenyl adenine for 14 days. The explants were transferred to basal medium without cytokinins for 3 weeks, and then to basal medium containing 0.05% activated charcoal. Elongating shoots were subcultured every 4 weeks on charcoal medium. Shoots, 10 mm in stem height, were rooted either in agar or sterilized peat/perlite (1:1). Up to 70% of the shoots formed roots when they were transferred to the latter, moistened with 1/2 strength Gresshoff and Doy medium containing 5 μM α-naphthaleneacetic acid. About 90% of the plantlets survived transfer to greenhouse conditions.

Development of two stage culture process by optimization of inorganic salts for improving catharanthine production in hairy root cultures of Catharanthus roseus

The effects of the concentrations of inorganic salts in Schenk and Hildebrandt (SH) medium on catharanthine production in hairy root cultures of Catharanthus roseus were investigated. The inorganic salt components could be categorized into four groups. The first group (nitrate) supported both the growth of and catharanthine production by hairy roots with incremental increases in the concentration. The second (ammonium and phosphate) yielded contradictory effects with respect to growth and production. The third (borate and molibdate) inhibited both growth and production, while the fourth (potassium iodide, sulfate, and iron) did not exhibit any significant effects. Through optimization of the concentrations of inorganic salts in the medium, a two stage process of hairy root cultures with different media for growth and production was developed which enabled us to enhance the volumetric yield of catharanthine up to 60.5 mg/ l. This productivity was 5.4 times higher than that of a one stage culture in the original SH medium.

Regenerating Plants from in Vitro Culture of Black Locust Cotyledon and Leaf Explants

A protocol to achieve efficient plant regeneration from juvenile black locust (Robinia pseudoacacia L.) explants is described. Direct adventitious shoots were induced from cotyledon explants on woody plant medium containing 22.2 μm BA and 0.4 μm 2,4-D. Shoots developed and new shoots were induced when the explants were transferred to medium without growth regulators. The effect of dark incubation on shoot regeneration from cotyledons indicated that 15 days of darkness resulted in a high regeneration frequency (91.7%). Adventitious shoot formation also was induced from sections of in vitro-derived leaves cultured in darkness on Murashige and Skoog medium supplemented with 4.4 μm BA and 24.6 μm IBA. A shoot regeneration frequency of 89% was obtained when explants were subcultured on a medium containing 4.4 μm BA and 0.5 μm IAA. Shoots were rooted on Schenk and Hildebrandt medium with or without IBA. Plantlets were acclimatized and grown in the greenhouse. Chemical names used: N -(phenylmethyl)-1H -purin-6-amine (BA); 2,4-dichlorophenoxyacetic acid (2,4-D); indole-3-acetic acid (IAA); indole-3-butyric acid (IBA).