Adventitious Shoot Formation Research Articles

A TWO-STAGE MICROPROPAGATION SYSTEM FOR CRANBERRIES

In an effort to accelerate breeding programs and to study somaclonal variation, a micropropagation system was devised for cranberries (Vaccinium macrocarpon). Using a factorial design, explants taken from greenhouse grown plants were placed on Anderson's medium containing different concentrations of 2ip' GA3, and IBA, with 4 cultivars tested over 3 subcultures. In other experiments, explant source, macro and micro salt formulations, and rooting treatments, were studied. Optimal multiplication and shoot quality occurred when single node explants taken from greenhouse grown plants were placed on Anderson's media containing 150 uM 2iP, 1.0 uM IBA and no GA3. Histological examinations indicate that initial response is axillary bud proliferation but upon subculture adventitious shoot formation may be possible. Proliferated shoots could be rooted ex vitro in plug trays under plastic tents and without hormone treatments. Optimal rooting occurred under high light conditions in a 1:1 (v:v) peat:sand mix. Plants were easily transplanted into the field in spring and will be evaluated by comparison to conventionally propagated material.

A Two-dimensional Electrophoretic Analysis of Protein Synthesis and Accumulation during Adventitious Shoot Formation in Somatic Tissue Cultures of Solanum carolinense L.

Adventitious shoot formation from tissue cultures of Solanum carolinense can be induced when stem segments are cultured on medium containing benzyladenine. When cultured on medium containing 2,4dichlorophenoxyacetic acid, stem explants produced only callus. Using two-dimensional gel electrophoresis, total soluble protein and protein labeled in vivo with [ 35 S]-methionine were extracted from regenerating and nonregenerating cultures and the gels or autoradiographs computer analyzed to identify different sets of proteins associated with development in vitro . Overall, the level of expression of 51 stained and 40 labeled proteins was altered, and it was shown that in vitro morphogenesis of stem segments of S. carolinense is associated with major quantitative changes in protein expression. Qualitatively, stagespecificpolypeptides related to callus formation and in vitro organogenesis were also identified.

An in vitro study of organogenesis in guayule ( Parthenium argentatum Gray)

The in vitro propagation of Guayule was achieved using shoot explants excised from aseptically germinated seedlings. Enganced adventitious shoot formation was obtained on Murashige and Skoog medium (MS) (half strength salts) supplemented with 0.5 mg 1 −1 6-benzylaminopurine (BA). Repeated subculturing of shoots resulted in a propagation rate of 10.67 ± 5.15 shoots per initial explant every 3 weeks. Optimal rooting (90%) occured on MS medium (three quarter strength) supplemented with 2 g 1 −1 m-inositol and 0.3 mg 1 −1 3-indolebutyric acid (IBA). Higher IBA concentrations resulted in a decrease in root length. A survival rate of 90% was obtained when the plantlets were hardened-off under greenhouse conditions.

Involvement of phytohormones in light-induced adventitious shoot formation of horseradish hairy root.

Involvement of phytohormones in light-induced adventitious shoot formation of horseradish hairy root.

In vitro development of adventitious shoots in Euonymus alatus (Celastraceae)

Mature embryos of Euonymus alatus (Thunb.) Sieb. were excised aseptically from seeds before or after cold treatment and germinated in light on half-strength Murashige and Skoog (MS) medium with 2% sucrose, 0.7% agar, naphthalene acetic acid (NAA) at 0.01 mg l −1 and benzylaminopurine (BAP) at 0.1–0.5 mg l −1. Hypocotyls of these seedlings were cut into 1–2-mm segments and induced to form adventitious shoots on the same medium. Large numbers of shoots, most bearing several pairs of leaf primordia, were produced per isolated hypocotyl segment. These shoots could be further multiplied by axillary bud release on medium containing 0–0.1 mg l −1 NAA and 0.0–0.3 mg l −1 BAP. Adventitious shoot formation began with profuse callus formation, originating in the epidermis of the hypocotyl. Vascular strands differentiated from callus parenchyma were observed extending into surface protuberances and, in older cultures, into adventitious shoot buds. Rooting was induced in vitro by culturing shoots on medium containing 0.4% agar and 3 mg l −1 indolebutyric acid (IBA). Rooted shoots were successfully transferred to vermiculite, hardened off and had the appearance of normal field-grown plants, including the characteristic winged periderm of this species.

Clonal propagation of guava ( Psidium guajava L.) from seedlings and grafted plants and adventitious shoot formation in vitro

Different parts of guava ( Psidium guajava L.) seedlings and the nodal segments of grafted plants were used as explants for in vitro culture. A high frequency (75–100%) of shoot regeneration was obtained from seedling hypocotyl, shoot tip and nodal segments cultured in MS medium with or without BA. The optimal BA Level for shoot tips and nodal segments is 0.1 mg l −1. Nodal segments form regenerated shoots were used for further multiplication; an average of 3.2 shoots segment −1 could be obtained after a period of 8 weeks in medium with 0.5 mg l −1 BA. Shoots were also regenerated from leaf explants. Regenerated shoots were rooted in basal medium with 100% rooting frequency and more than 90% of the plantlets were successfully established in soil.

ADVENTIVE ORGANOGENESIS FROM SOMATIC TISSUE CULTURES OF SOLANUM CAROLINENSE: ORIGIN AND DEVELOPMENT OF REGENERATED PLANTS

The development of adventitious shoot formation from cultured somatic tissues of Solanum carolinense was studied using light microscopy. For purposes of comparison, callus initiation and proliferation were also followed. When stem segments of this plant were cultured on medium supplemented with 6 mg/l benzyladenine (BA), cell division was first observed after 48 hr in the external phloem and inner cortex of the segments. This division activity gave rise to meristematic zones which subsequently formed shoot primordia within 6 days of culture. While organogenic potential appears to be limited to specific regions, all tissues of the explant were capable of callus formation when cultured on medium containing 3 mg/l 2,4‐dichlorophenoxyacetic acid (2,4‐D). In conjunction with a previous study, it appears that unlike somatic embryogenesis in this species, organogenesis does not require an intermediate callus phase for differentiation to occur.

Adventive Organogenesis from Somatic Tissue Cultures of Solanum carolinense: Origin and Development of Regenerated Plants

The development of adventitious shoot formation from cultured somatic tissues of Solanum carolinense was studied using light microscopy. For purposes of comparison, callus initiation and proliferation were also followed. When stem segments of this plant were cultured on medium supplemented with 6 mg/l benzyladenine (BA), cell division was first observed after 48 hr in the external phloem and inner cortex of the segments. This division activity gave rise to meristematic zones which subsequently formed shoot primordia within 6 days of culture. While organogenic potential appears to be limited to specific regions, all tissues of the explant were capable of callus formation when cultured on medium containing 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). In conjunction with a previous study, it appears that unlike somatic embryogenesis in this species, organogenesis does not require an intermediate callus phase for differentiation to occur.

Adventitious root and shoot regeneration in cultured explants of mature black spruce

Embyronic shoots with crown excised from vegetative buds of 15–35-year-old black spruce [ Picea mariana (Mill.) B.S.P. were induced to form adventitious roots from either the cortex at the shoot base or the axillary region of leaf primordia. The roots were initiated in one-half-strength Gresshoff and Doy (Planta, 107 (1972) 161) medium after the embryoic shoots had been cultured on the medium supplemented with 2,4-dichlorophenoxyacetic acid and naphthaleneacetic acid for 4 weeks or less. Prolonged incubation on the medium with growth regulators resulted in differentiation of adventitious roots on leaf primordia. Formation of adventitious shoots occured when embryonic shoots were cultured on one half or full strength Gresshoff and Doy medium supplemented with benzylaminopurine and naphthaleneacetic acid for 4 weeks or less before being transferred onto the respective medium without any growth regulators. Adventitious shoots often differentiated from the axillary region of leaf primordia on the embryonic shoots. Some leaf primordia differentiated directly into adventitious shoots and others produced calli before differentiation of the shoots. Culture on the medium with growth regulators for more than 4 weeks increased callus formation and decreased adventitious shoot regeneration on embryonic-shoots.

Adventitious shoot formation from embryonic explants of red pine (Pinus resinosa)

Adventitious shoots were induced from excised embryos of Pinus resinosa Ait, on half‐strength Le‐Poivre (LP) medium containing 1–70 μMN6‐benzyladenine (BA). At lower concentrations of BA, only 2–3 shoot primordia (from as many as 22 formed per embryo) developed into shoots when subcultured onto medium containing 0.5% activated charcoal. Concentrations of 10 to 70 μM of BA produced significantly higher numbers of shoot primordia and most of them developed into shoots. Ten to 17 day culture on medium containing 10–25 μM BA proved optimal for maximum adventitious shoot production. Less than three days of incubation on the cytokinin medium did not stimulate the formation of adventitious shoots. Twenty‐four day culture on the same medium produced several shoots, but most of them failed to develop normally and formed callus. Coconut milk (0.1–5% v/v) inhibited adventitious shoot formation. Using optimal conditions, seeds from 11 open‐pollinated selected trees were compared to test for genetic differences in the potential to produce adventitious shoots from embryos. No significant differences were observed with regard to the shoots produced per embryo among the different seed collections. More than 200 plants produced through this technique were tested for variation in several isozymes by electrophoresis. No variations were observed.

* IN VITRO CULTURE OF HIGHER PLANTS AS A TOOL IN THE PROPAGATION OF HORTICULTURAL CROPS

In vitro culture of higher plants is the culture, under sterile conditions, of plants, seeds, embryos, organs, explants, tissues, cells and protoplasts on nutrient media. This type of culture has shown spectacular development since 1975, resulting in the production and regeneration of viable individuals of many plant species. In addition, since 1980 there has been an explosion in the development of genetic manipulation and biotechnology. Various types of cultures and their applications are discussed: embryo culture, orchid seed culture, meristem culture, cloning of crops (single-node culture, meristem culture, axillary branching, adventitious shoot formation, callus culture, single cell and protoplast culture), anther and microspore culture, culture of ovules and excised flowers, and protoplast culture. Vegetative propagation in vitro (also called micropropagation) will be dealt with in greater detail since it has yielded results which are of enormous importance in horticulture. A recent survey in the Netherlands has shown that the number of commercial tissue culture laboratories in this country has increased from 28 in 1983 to 50 in 1986; the total number of plants cloned in vitro increasing from 21 to 43 million over the same period. The widespread use of in vitro cloning is an indication of its many advantages. However, there are also disadvantages. The criteria desired for the success of in vitro cloning can be itemized as follows: no induction of mutations; the starting material must be disease-free, or disease-free material must be produced by meristem culture; the transfer from test tube or container to soil should not be too difficult; the regeneration ability should not be lost; the technique should be economically viable and not too complicated (synthetic seeds!), otherwise it will be rejected. Woody species (trees and shrubs) are far more difficult to clone in vitro than herbaceous plants; the reasons for the differences are given. In vegetative propagation and propagation by seeds, in vitro culture can also be a tool to: obtain disease-free plants; transport disease-free plant material; increase the genetic variation (by somaclonal variation and genetic manipulation); induce haploids; separate chimeras; obtain tetraploids; store plant material in gene banks where it can be kept disease-free under conditions which limit growth and development.

Determinate Floral Buds of Plantain (Musa AAB) as a Site of Adventitious Shoot Formation

Determinate Floral Buds of Plantain (Musa AAB) as a Site of Adventitious Shoot Formation

In vitro adventitious shoot formation on mature-phase leaves and petioles of Liquidambar styraciflua L.

Adventitious shoots were induced to form on leaves and petiole segments of mature-phase Liquidambar styraciflua L. Shoot organogenesis occurred directly, without the formation of a distinct callus stage, and well-defined shoots were visible in 6–9 weeks. Prolific shoot production was supported by Woody Plant Medium supplemented with relatively high levels of benzyladenine (2.5 mg/l). Changes in benzyladenine concentration and the addition of 0.1 mg/l naphthaleneacetic acid to the medium altered the relative abundance of shoots on various parts of a leaf. Shoot formation occurred most frequently at or near breaks in major vasculature. Wounding of the leaves by slashing across the lamina and vasculature significantly increased the total number of shoots formed per explant and also altered the pattern of organogenesis. Differences in organogenic response were seen between the cultivars ‘Moraine’ and ‘Variegata’. Shoots derived from leaves were easily rooted and acclimated to greenhouse conditions. Three new variegation types arose in vitro as a result of adventitious shoot formation on ‘Variegata’ leaves.

Callus formation and plant regeneration capacity of apple embryonic axes and cotyledons in relation to seed dormancy

Embryonic axes and cotyledons isolated from dormant and 60-day stratified apple seeds showed the ability of dedifferentiation and organogenesis during the culture in vitro. Dormant embryonic axes dedifferentiated to callus tissue more easily than dormant cotyledons. Similarly, adventitious shoot formation on dormant embryonic axes was higher than on dormant cotyledons. In stratified organs, however, the ability for callus and shoot formation did not differ. Regeneration of shoots from calli was higher for embryonic axes-derived callus than for cotyledon-derived callus. Together with stratification, the frequency of shoot regeneration increased for callus originating from axes and markedly decreased for callus obtained from cotyledons. Calli with initiated shoots multiplicated independently of the organ used for the initial culture. The growth of dormant organs' shoots, unlike stratified organs' shoots, showed some morphological anomalies. Rooting of both cultured organs, dormant or stratified, was poor. The only factor that influenced root formation was the auxin present in the culture medium.

An improved medium for adventitious shoot formation and callus induction in Beta vulgaris L. in vitro

Six sugarbeet (Beta vulgaris L.) lines (GWI-248, SPB-11, MonoHy 55, SMS-1, EL45 and FC607) were tested for regeneration. Shoot cultures were initiated in vitro from naked, sterilized embryos obtained from mature seed. Excised petioles from cultured shoots were plated on Gamborg's B5 medium and four modified Murashige and Skoog (MS) media. A medium containing MS inorganic salts supplemented with 0.4 mg/1 N(6)-benzyladenine, 0.1 mg/1 indole-3-butyric acid, ten vitamins and six amino acids, termed RV, was superior for both adventitious shoot and callus formation. Callus was observed only on RV medium and only on petioles that did not develop adventitious buds directly. Rooting of regenerated shoots and development of complete plants was accomplished by transfer to Gamborg's B5 medium with 5 mg/l indole-3-butyric acid as the sole phytohormone. The complete process of regeneration through adventitious shoot production took from 4 to 6 weeks from explants to rooted plants. The callus that formed on nonorganogenic petioles was regenerative when transferred to fresh RV medium. Regeneration from callus occurred mainly by shoot organogenesis but also by somatic embryogenesis at a low frequency.

Influence of macronutrient composition, TIBA and dark treatment on shoot formation and nitrogen content in petiole explants ofSenecio × hybridus

SummaryThe content of ammonium, nitrate and potassium was varied in the macronutrient solutions intended for formation of adventitious shoots from petiole expiants of Senecio × hybridus. The other components of the macronutrients were according to Murashige and Skoog (1962). The largest number of expiants which formed shoots was obtained when the nitrogen concentration in the Murashige-Skoog solution was lowered from 60 to 30 mM and the potassium concentration from 20 to 15 mM. Addition of 1.0 μM TIBA to the medium as well as the standard addition of 4.44 μM BAP and 28.5 μM IAA favoured shoot formation. Even growth in darkness for two weeks immediately after expiant excision increased shoot number. The nitrogen content in the tissue decreased as the nitrogen concentration in the medium decreased, although an increased concentration in the medium from 60 to 75 mM did not increase the nitrogen content in the tissue. When the potassium concentration was changed from 20 to 15 mM, in a medium with 30 mM nitrog...

Effects of Phenolic Compounds on Tobacco Callus and Blackberry Shoot Cultures

Abstract The effects of catechol, juglone, phloridzin, phloroglucinol, and tannic acid were tested on tobacco callus growth and on axillary shoot proliferation and rooting of blackberry. Juglone (500 μM) promoted tobacco (Nicotiana tabacum L. ‘McNair 944’) callus growth, whereas catechol and phloroglucinol (500 μM) increased adventitious shoot formation, but not callus growth. Tannic acid increased the frequency of vitreous shoots. No phenolic compounds stimulated axillary shoot proliferation in blackberry (Rubus sp. ‘Dirkson Thornless’), and phloroglucinol (1 mM) inhibited shoot elongation. Auxin increased rooting of blackberry shoots irrespective of the presence or absence of incorporated phenols. On shoots derived from proliferation media void of phenols, root number and percent rooting were greater if they were treated with the phenolic auxin P-ITB compared to IBA. In contrast, shoots originating from proliferation media containing phenols rooted the same with both auxins. Chemical names used: 1,2-benzenediol (catechol); 5-hydroxy-1,4-naphthalendedione (juglone); 1-[2-(β-D-glycopyranosloxy)-4,6-dihydroxyphenyl-3-[4-hydroxyphenyl)-1-propanone (phloridzin); 1,3,5-benzenetriol (phloroglucinol); 1H-indole-3-butanoic acid (IBA); N-(phenylmethyl)-1H-purin-6-amine (BA): phenyl indole-3-thiolobutyrate (P-ITB); (1α,2β,4aα,4bβ,10β)-2,4a,7-trihydroxy-1-methyl-8-methylenegibb-3-ene-1, 10-dicarboxylic acid 1,4a-Iactone (GA3).

Summary and Future Direction: Chemical Regulation in Tissue Culture

Abstract This series of papers has expanded upon Skoog's milestone discovery regarding the roles of cytokinins and auxins in morphogenesis of in vitro systems. One of the effects of adenine-based cytokinins in culture systems is the formation of adventitious shoots or the expression of axillary shoots. This symposium reported that a cytokinin response also can be observed by using phenylurea-based cytokinins. Two phenylurea compounds, thidiazuron and 4PU–CI, were illustrated as having strong cytokinin activity, equivalent to or surpassing the adenine-based compounds. One of the effects of these diphenylureas is the formation of autonomous callus. These observations were used to speculate on the mode of action of the phenylureas as being stimulation of endogenous cytokinin biosynthesis (or metabolism), resulting in altered cytokinin levels. Other studies indicate that the phenylureas and adenine cytokinins may have similar action sites, hence similar effects.

The effect of supplemental irradiation with different light sources on growth and flowering of gloxinia ( Sinningia speciosa (Lodd.) Hiern)

The relative efficiency of a warm white fluorescent lamp, a high-pressure metal halide lamp, a high-pressure sodium lamp and a low-pressure sodium lamp on growth and flowering of three cultivars of Sinningia speciosa (‘Burgdorf’, ‘Gerda Lodder’, ‘Peace’) has been studied under winter conditions at latitude 60°N. The light sources were compared at three irradiance levels (10, 14 and 18 W m −2 in the wavelength range 400–1000 nm). The growth rate and growth habit were markedly affected by both light source and irradiance level, and specific cultivar reactions were recorded. The differences recorded between light sources in rate of development, dry weight production, plant diameter and adventitious shoot formation were in general more pronounced at lower than at higher irradiance levels.

Improved bunt inoculation of wheat utilizing adventitious shoots

A procedure is described of injecting inoculum of dwarf or common bunt at the bases of the internodes of wheat. Physical stimulation by wounding of the intercalary meristem located above the nodes resulted in the formation of adventitious shoots. If inoculum of dwarf or common bunt is injected at the time of stimulation, the spike of the resulting shoot is usually infected. This procedure shortened the period between teliospore germination and production of sori, gave a high infection rate, and induced several infected spikes on one primary stem.