In vitro adventitious root induction in Antirrhinum majus L.

A broadly applicable method for the successful induction of root systems in a number of cultivars of A. majus has been determined. This involves a double filter-paper bridge with a liquid medium for root induction and allows the transfer of culture-grown plantlets to a glasshouse environment with minimal disturbance to the plant as a whole. 100% survival of transferred plantlets has been achieved with the inclusion of a few simple precautions upon shoot transfer and during the initial stages of plant establishment in vivo.     

Induction of adventitious shoots and tetraploids in Antirrhinum majus L. by treatment of antimitotic agents in vitro without plant growth regulators

We examined the effects of five antimitotic agents using Antirrhinum majus L. ‘Maryland True Pink’ on the induction of adventitious shoots resulted in increase of frequencies of chromosome doubling without plant growth regulators. Seeds were treated in vitro with 0, 16.5, 32.9, 65.8, 131.6, or 263.2 µM oryzalin (ORY), amiprofos-methyl (APM), butamifos (BUT), or propham (IPC) or 800, 1,600, 3,200, 6,400, or 12,800 µM colchicine (COL) for 7 day. ORY, COL and APM promoted induction of adventitious shoots on the hypocotyls at maximum frequencies of 57.6% with 16.5 µM ORY, 5.6% with 800 µM COL and 88.8% with 131.6 µM APM. ORY and COL also induced adventitious shoots on the epicotyls adjacent to the cotyledons, particularly at high concentrations, with a maximum frequency of 26.0% at 12,800 µM COL. APM treatment increased frequencies of tetraploids from 0.0 to 93.1%, with a positive correlation between the frequency and concentration. By contrast, ORY and COL induced tetraploids at frequencies of 16.0 to 54.6% and 4.0 to 59.4%, respectively, with peaks at both low and high concentrations of each. Correlation analysis revealed that frequencies of adventitious shoot formation could be useful as an index for the induction of tetraploids. These results showed that three of the antimitotic agents tested induced both adventitious shoot and tetraploid without plant growth regulators, indicating that antimitotic action may play a common role in the induction of adventitious shoot.     

Regeneration of plants from Antirrhinum majus L. callus

Somaclone production in Antirrhinum majus plants by regeneration of plants from callus cultures has been achieved using three types of explant tissue. Regeneration from mature stem internode-derived callus was extremely poor. Callus derived from seedling shoot tips could be induced to form new shoots in six of seven cultivars tested. Regeneration was achieved in all seven cultivars when callus was produced from segments of hypocotyl and was most effective using agar-solidified medium containing 0.25 mgl^-1 naphthoxyacetic acid + 10% coconut milk. In this case, five of the cultivars produced shoots directly, one produced leaves from the petioles of which new shoots emerged, and one regenerated plants chiefly through the production of embryoids.     

Multiplication of Antirrhinum majus L. by shoot-tip culture

Nine varieties of Antirrhinum majus L. have been used in a study of in vitro multiplication of plants using shoot-tip culture. Acceptable multiplication rates were obtained in several media with only variety Victory showing significantly lower rates of shoot production. Wounded shoots of this variety produced callus in the absence of added auxin and some of this callus produced prolific roots.     

High-frequency induction of adventitious shoots from hypocotyl segments ofLiquidambar styracifiua L. by thidiazuron

The effects of thidiazuron (TDZ) on adventitious bud and shoot formation from hypocotyl segments of sweetgum (Liquidambar styracifiua) were tested alone and in combination with 2,4-dichlorophenoxyacetic acid (2,4-D). The combination of 1 mg/1 TDZ with 0.01 mg/l 2,4-D resulted in the highest frequency of bud production. Lower concentrations of TDZ stimulated shoot production, generating the most shoots at 0.1 mg/1 TDZ with 0.01 mg/1 of 2,4-D. Inhibition of shoot elongation by TDZ was overcome by transferring shoot cultures to a shoot proliferation medium lacking TDZ or containing naphthaleneacetic acid and benzyladenine in addition to TDZ. Shoot production in liquid culture was significantly greater than that in solid culture. Comparisons of in vitro and ex vitro rooting of the adventitious shoots demonstrated that ex vitro rooting produced plants with faster growth rates and more extensive root systems.Abbreviations BA Benzyladenine - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - 2,4-D 2,4-dichlorophenoxyacetic acid     

The Ultracytochemical Localization of ATPase Activity in the Ovules of Antirrhinum majus L.

The ultracytochemical localization of ATPase activity was carried out by the method of lead precipitation in the ovules of Antirrhinum majus L. No ATPase activity is observed in the egg apparatus, but some in the polar nuclei, cytoplasm and plasma membrane of the central cell. Between the embryo sac wall and the cuticle surrounding it, there is a gap where some filamentand vesicle-like structures were demonstrated by conventional staining method, and much of ATPase activity is found there. At the chalaza of the ovule, a lot of ATPase particles are found irt the nuclei, plasma membranes and the thick and loose wall of the hypostase cells. The particles of ATPase in the hypostase and those in the gap surrounding embryo sac are continuously distributed through the intervals of the cuticle at the chalazal end of the embryo sac. Some of ATPase particles are found on the plasma membranes and plasmadesmata of integument ceils, noticeably much more in the nucleoplasm of the integumentary tapetum. According to the ATPase distribution pattern in the ovules, we suggest that the function of the integumentary tapetum and hypostase is secretion, and that the gap surrounding the embryo sac may be an apoplastic ehannal for nutrient flow into the embryo sac.     

Induction of adventitious buds in cultured petal explants ofChelidonium majus L.

Petal explants ofChelidonium majus L. (Papaveraceae) formed noteworthy adventitious buds without any intermediate callus when cultured under appropriate conditions. Bud formation was favored by combinations of 1–2 mg/l indoleacetic acid (IAA) and/or 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1–0.5 mg/l kinetin (K). In the present study, neither bud formation nor callus formation occurred in cultures of excised leaves. A histological study revealed that adventitious bud formation occurred only in single epidermal layers of petals, while several subepidermal parenchyma layers did not join in its formation. Activation zones arising from the epidermis underwent intense cell divisions to initiate buds on the epidermal surface. These buds later turned green in color, developing into shoots which eventually grew into plantlets after root formation.     

Isolation and Cultural Behavior of Pollen Tube Subprotoplasts in,Antirrhinum majus L.

Large numbers of subprotoplasts were isolated enzymatically from pollen tubes of Antirrhinum majus L. When these subI)rotoplasts, either nucleate or enucleate, were cultured in D2 liquid eulture medium, each formed a thick cell wall and germinated a pollen tube like strueture which also deposited a thick wall, except at the tip of the tube. Tube growth was accomparied by a continuous movement of the mass of cell inelusion in this tube to the tip. Rupture of the naked tip oeeurred within one to six days releasing the mass of cell inelusion in the tube into the culture medium. The faet that both nucleate and enneleate subprotoplasts show the same cultural behavior eharaeteristie of the gene expression of a normal pollen tube demonstrates the presence of presynthesized mRNA in the germinated tubes.     

In vitro induction of tetraploid plants from diploid Zizyphus jujuba Mill. cv. Zhanhua

Tetraploid plants of Zizyphus jujuba Mill. cv. Zhanhua were obtained with in vitro colchicine treatment. Shoot tips from in vitro-grown plants were treated with five different concentrations of colchicine (0.01, 0.03, 0.05, 0.1, 0.3%) in liquid MS medium (Murashige and Skoog 1962), and shaken (100 rpm) at 25 °C in darkness for 24, 48, 72 or 96 h, respectively. Tetraploids were obtained at a frequency of over 3% by using 0.05% colchicine (48 h, 72 h) and 0.1% colchicine (24 h, 48 h) treatment as determined by flow cytometry. Cytological and morphological evidence confirmed the results of flow cytometric analysis. The chromosome number of diploid plants was 24 and that of tetraploid plants was 48. The stomata sizes of tetraploid plants were significantly larger than those of diploid plants, while the frequency of stomata were reduced significantly. Similarly, the chloroplast number of guard cells of tetraploid plants increased significantly. The selected tetraploid plants were grafted onto mature trees of Z. jujuba Mill. cv. Zhanhua in the field, resulted in thicker stems, rounder and succulent leaves, larger flowers and a delay in florescence time (3–4 days later) than diploid plants.     

Variation in the Photoperiodic Control of Flowering of Two Cultivars of Antirrhinum majus L.

The susceptibility to photoperiodic induction of an early (PinkIce) and a late (Orchid Rocket) flowering variety of Antirrhinummajus was investigated. At various times during developmentplants of both varieties were subjected to 1, 2, 3 or 4 long-daycycles. The early variety became increasingly more responsiveto long-days while the late variety showed no positive responsethroughout the course of the experiment. One hour light-breaksgiven midway through the 16-h dark period evoked a decreasein the leaf number of Pink Ice plants. The leaf areas of bothvarieties increased to levels equivalent to long-day grown plants.The net CO₂ uptake of plants grown in light-breaks increasedin proportion to the increase in leaf area. However the specificuptake of CO₂ per unit area was similar for light-break andshort-day grown plants but was substantially lower than thelong-day plants. Suggestions are made to explain the differencesin photoperiodic response of the two varieties in terms of variationin assimilation rate.     

The diversity and ontogenetic changes of phyllotaxis in wild-types and two leaf form mutants of Antirrhinum majus L.

We have analysed the phyllotactic patterns of the main shoot in vegetative and generative phases of growth in wild type and mutant plants of Antirrhinum majus L. Wild types 'Sippe50' and 'W l08' were compared to mutants grminifolia and phanlastica . The normal vegetative phyllotaxis of the wild type plants is decussate, but the inflorescence phyllotaxis is spiral and of the Fibonacci type. The phyllotaxis patterns of the mutants differ strongly from that of the wild type. Besides decussate phyllotaxis, whorls of three or four elements as well as spiral patterns in vegetative phase were observed. The vegetative phyllotaxis in mutants is ontogenetically unstable with frequent transitions between patterns, including the reversion of chirality of spiral phyllotaxis. The number of transitions per plant was larger in graminifolia than in phantastica . The inflorescence phyllotaxis was more stable and occasional non-typical phyllotaxis patterns finally transformed to a Fibonacci pattern. The results suggest a possible role of genetic factors in determining the regularity of spatial arrangement of organs.     

The effects of antibiotics and their breakdown products on the in vitro growth of Antirrhinum majus

Summary The effects of various antibiotics on the development of hypocotyls of Antirrhinum majus in tissue culture have been studied. The penicillins, carbenicillin and penicillin G, have been shown to stimulate callus growth, have little impact on shoot production and may stimulate root formation. The cephalosporins, cephotaxime and cephalosporin, have no effect on callus production and reduce shoot and root formation. HPLC, GC and GC-MS analyses have shown that concentrations of carbenicillin and penicillin G, commonly used in plant tissue culture, break down to give physiologically active levels of the auxin phenylacetic acid. This offers a mechanism for the stimulation of growth caused by these two antibiotics.Abbreviations Amp ampicillin - 6APA 6 aminopenicillanic acid - BAP benzyl amino purine - Cb carbenicillin - Cb_n and Cb_o 1 month and 1 year old samples respectively of carbenicillin stored at 4°C as powder - Cph cephalothin - Cx cephotaxime - IAA indoleacetic acid - MS Murashige and Skoog medium - NOA -naphthoxyacetic acid - PAA phenylacetic acid - PenG benzyl penicillin - PMA phenylmalonic acid     

In vitro morphogenetic response and distribution of endogenous plant hormones in hypocotyl segments of snapdragon (Antirrhinum majus L.)

Snapdragon seedlings, 20 mm in length, were cut into 5 segments from the cotyledon to the root, which were cultured in vitro on hormone-free MS medium. Adventitious shoot formation was highest in the basal hypocotyl segments with stimulation by the addition of BA. Endogenous cytokinins were higher in the basal hypocotyl segments than in the two upper hypocotyl segments, whereas auxin content was higher in the two upper than in the basal hypocotyl segments. Ratios of cytokinins to auxin were also the highest in the basal hypocotyl segment. A general principle in in vitro culture that a high concentration of cytokinin and a low concentration of auxin promotes the induction of shoot morphogenesis was confirmed from measurements of endogenous growth regulator concentrations.     

Efficient shoot regeneration from hairy roots of Antirrhinum majus L. transformed by the rol type MAT vector system

 Eleven independent GUS-positive hairy roots were induced by co-cultivation of leaf explants of Antirrhinum majus L. with Agrobacterium tumefaciens strain GV2260 containing the rol type MAT vector pNPI702. The MAT vector pNPI702 possesses a GUS gene under the 35 S promoter and a removal element in which the 7.6-kb DNA fragments containing the rolA, B, C and D genes and recombinase gene with a 35 S promoter are located between two directly oriented recombination site sequences. A total of 326 adventitious shoots regenerated from 11 independent hairy root lines cultured on 1/2MS medium without plant growth regulators at 25  °C under a 16/8 h (day/night) photoperiod after 8 weeks of stock-culture of hairy roots and 4 weeks of culture of the green segments of hairy roots. Regenerated plants showed either a normal or dwarf morphology. GUS activity was observed in the hairy roots and regenerated shoots. The presence of the GUS gene in the regenerated, morphologically normal plants was confirmed by PCR analysis. Received: 28 February 2000 / Revision received: 18 August 2000 / Accepted: 22 August 2000     

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 N⁶-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.Abbreviations BA N⁶-benzyladenine - IBA indole-3-butyric acid Contribution from Missouri Agricultural Experiment Station. Journal Series No. 10394. University of Missouri, Columbia, MO 63873, USA Mention of trade or company name does not constitute a guarantee or warranty of the product by University of Missouri-Columbia or U.S.D.A. Agricultural Research Service and does not imply their approval to the exclusion of other products that may be suitable.     

In vitro induction and identification of tetraploid plants of <Emphasis Type="Italic">Paulownia tomentosa</Emphasis>

Polyploidization is a major trend in plant evolution that has many advantages over diploid. In particular, the enlargement and lower fertility of polyploids are very attractive traits in forest tree breeding programs. We report here a system for the in vitro induction and identification of tetraploid plants of Paulownia tomentosa induced by colchicine treatment. Embryonic calluses derived from placentas were transferred to liquid Murashige and Skoog (MS) medium containing different concentrations of colchicine (0.01, 0.05, or 0.1%) and incubated for 24, 48, or 72 h on an orbital shaker at 110 rpm. The best result in terms of the production of tetraploid plantlets was obtained in the 48 h + 0.05% colchicine treatment, with more than 100 tetraploid plantlets being produced. The ploidy level of plantlets was verified by chromosome counts, flow cytometry, and morphology. The chromosome number of tetraploids was 2n = 4x = 80 and that of diploid plantlets was 2n = 2x = 40. The relative fluorescence intensity of tetraploids was twofold higher than that of diploids. The tetraploid and diploid plantlets differed significantly in leaf shape, with those of the former being round and those of the latter pentagonal. The mean length of the stomata was longer in tetraploid plants than diploid plants, and stomatal frequency was reduced with the increased ploidy level. The tetraploids had large floral organs that were easily distinguishable from those of diploid plants.     

In vitro induction of tetraploid plants from a diploid Japanese pear cultivar (Pyrus pyrifolia N. cv. Hosui)

Tetraploid plants of a Japanese pear cultivar (Pyrus pyrifolia N.) were induced using an in vitro colchicine treatment. Proliferating shoots were transferred to a shoot proliferation medium (SPM) containing 0.1% or 0.01% colchicine, incubated for 1, 2, 4 or 8 days, then transferred to fresh SPM. The ploidy level of the colchicine-treated individuals was analysed by flow cytometry. Four months after colchicine treatment four mixoploids were selected and cultured on SPM for a further 5 months. The ploidy level of the proliferated shoots derived from selected mixoploids was analysed, and five tetraploid shoots were selected. The selected tetraploid shoots were rooted, and potted plants were grown in a greenhouse. The stomata of tetraploid plants were found to be longer than those in diploid plants.     

In vitro induction of multiple shoots and plant regeneration in cotton (Gossypium hirsutum L.)

Induction of multiple shoots in cotton (Gossypium hirsutum L. cv. Anjali-LRK 516) has been achieved with cotyledonary nodes devoid of cotyledons and apical meristems. Explants from 35-day-old seedlings yielded the maximum number of shoots (4.7 shoots/explant) using Murashige and Skoog (MS) basal medium supplemented with 6-benzylaminopurine and kinetin (2.5 mg/1 each). Explants from 35-day-old seedlings raised in glass bottles produced a higher number of multiple shoots (8.3 shoots/explant) than those grown in glass tubes and cultured on the same shoot induction medium. Elongation of multiple shoots was obtained on liquid or agar MS basal medium without phytohormones. In vitro shoots were rooted on half-strength agar-solidified MS basal medium or with 0.05 or 0.1 mg/1 naphthaleneacetic acid. Hardening and survival of tissue culture plantlets was 95% under greenhouse conditions.Abbreviations BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - MS Murashige and Skoog medium - NAA -Napthaleneacetic acid