Tissue culture and plant regeneration of watermelon (Citrullus vulgaris Schrad. cv. Melitopolski)

Plants were regenerated from cotyledon and hypocotyl explants of watermelon (Citrullus vulgaris). The explants were cultured on a Murashige and Skoog's basal nutrient medium supplemented with auxin, cytokinin and auxin-cytokinin combinations. Green healthy nodular and compact callus was obtained in medium containing naphthalene acetic acid and benzylaminopurine. Shoot differentiation and root differentiation from the cotyledon and hypocotyl after callus formation in different media containing benzylaminopurine or naphthalene acetic acid, respectively. Shoot formation required benzylaminopurine. Kinetin proved ineffective in inducing shoot buds or shoots. Root differentiation occurred in a medium containing naphthalene acetic acid or indole acetic acid. There was a greater proliferation of roots on medium supplemented with naphthalene acetic acid. The regenerated shoots developed roots when transferred to medium containing naphthalene acetic acid and complete plantlets could be transferred to soil for further growth.Abbreviations BAP 6 Benzylaminopurine - NAA -Naphthalene acetic acid - MS Murashige and Skoog's medium - IAA Indole acetic acid - KN Kinetin     

Rapid differentiation of tracheary elements in cultured explants of Jerusalem artichoke

the culture of Jerusalem artichoke (Helianthus tuberosus L.) tuber explants on filter paper discs moistened with liquid medium resulted in rapid and consistent xylem differentiation. The number of tracheary elements increased in discrete steps, the first at 48 h with a second at 56–58 h, following partially synchronous mitoses at 20 and 30 h. Factors favouring xylem cell differentiation were optimum levels of both an auxin and a cytokinin, low medium nitrogen concentrations, small volumes of medium, and high culture temperatures. A cell counting method employing Feulgen-stained nuclei and suitable for quantifyings small numbers of immature tracheary elements is described.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA -naphthalene acetic acid - BAP benzylaminopurine - GA₃ gibberellic acid     

High efficiency plant regeneration from cotyledons of watermelon (Citrullus vulgaris Schrad)

Cotyledons of various ages from seedlings of eight watermelon (Citrullus vulgaris) cultivars were cultured on MS medium supplemented with different combinations of phytohormones. High frequency shoot regeneration (60.0–92.0%) was induced from 5-day-old cotyledons of cultivars cultured on MS medium containing 5.0 mg/l 6-benzylaminopurine (BA) and 0.5 mg/l indole-3-acetic acid (IAA). Multiple shoot buds elongated on MS medium containing 0.2 mg/l kinetin (KT) and 5–10 shoots per expiant could be recovered depending on the cultivars. Elongated shoots rooted on MS medium with 0.1 mg/l -naphthalene acetic acid (NAA). Zeatin riboside (ZT) had a similar efficiency as BA in shoot induction, and it was significantly more functional than 2-isopentenyladenine (2iP) or kinetin (KT). Cotyledons from 5-day-old seedlings were the most responsive to shoot induction.Abbreviation BA 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - 2iP 2-isopentenyladenine - KT kinetin - MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - ZT zeatin riboside     

Regeneration of Arabidopsis callus in vitro

This paper reports on an easy and reproducible method of regenerating Arabidopsis plants from callus culture. A combination of 6-benzylaminopurine (BAP) and -naphthalene acetic acid (NAA) in a Murashige and Skoog's (MS) based medium gives a high percentage of shoot formation in several genotypes.     

In vitro propagation of Lilium testaceum and structural investigation of the storage β-1,4-glucomannan

Bulblet and callus cultures of Lilium testaceum were initiated in vitro from bulbscales. Continous propagation of the bulblet cultures was achieved on a modified Murashige and Skoog agar medium containing 1-naphthalene acetic acid (0.1 mg/l) and kinetin (0.1 mg/l) as phytohormones. The in vitro grown bulbs synthesized large quantities of storage ß-1,4-glucomannans (mannose: glucose = 73; molecular weight = 200 kd) with an identical structure to the glucomannans from the in vivo grown bulbs. Higher 1-naphthalene acetic acid concentrations (1 mg/l) resulted in increased callus formation. Liquid suspension cultures derived from callus exhibited only small amounts of reserve glucomannans.Abbreviations DEAE 2-(diethylamino)ethyl - GF growth factor - GM glucomannan - GPC gel permeation chromatography - IAA indole-3-acetic acid - IEC ion exchange chromatography - MS Murashige and Skoog - MW molecular weight - MWCO molecular weight cut off - NAA 1-naphthalene acetic acid - NMR nuclear magnetic resonance - PVP polyvinylpyrrolidone     

Taxus callus cultures: Initiation,growth optimization,characterization and taxol production

Callus was induced from Taxus baccata cv. Repandens Parsons ex Rehd., T. brevifolia Nutt., T. cuspidata Sieb. & Zucc., and T. x media cvs. Hicksii and Densiformis Rehd. using different concentrations of 2,4-d-(2,4-dichlorophenoxyacetic acid), IBA (indole-3-butyric acid), or NAA -naphthalene acetic acid in combination with kinetin. All cultures grew slowly following the first subculture, and a majority turned brown and ceased growth within the next six to twelve months. The callus cultures which lived, continued to grow very slowly for one to two years before the growth rate improved. Initiation of roots and shoot primordia-like structures occurred on some cultures maintained in the dark, and 16 h light/8 h dark, respectively. A fast-growing, habituated callus line (CR-1) derived from T. x media Rehd. cv. Hicksii was established from callus initiated in 1986. Supplementing the medium with casein hydrolysate and both fructose and glucose enhanced the growth rate. A great deal of heterogeneity was found among and within the callus, with respect to the amount of taxol produced. The callus exhibited levels of taxol ranging from 0.1 to 13.1 mg kg^-1 (0.0001 to 0.0131%) on a dry weight basis. Overall, the older brown-colored callus produced more taxol than the younger pale yellow-colored callus. The presence of taxol in callus samples was established by high performance liquid chromatography, its biological activity confirmed by a microtubule-stabilizing bioassay and its structure confirmed using one-and two-dimensional ¹H and ¹³C nuclear magnetic resonance spectroscopy.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - kinetin 6-furfurylaminopurine - 2iP 6-(,-dimethylallylamino)purine     

The effect of auxin concentration on cytokinin stability and metabolism

The stability of [³H]zeatin riboside supplied to freshly excised tobacco pith explants was found to be inversely related to -naphthaleneacetic acid concentration in the incubation medium. At higher concentrations of -naphthaleneacetic acid greater breakdown of [³H]zeatin riboside was indicated by higher levels of degradative metabolites (adenine, adenosine and adenosine nucleotides) formed. This auxin effect on cytokinin metabolism appears to be mediated, at least in part, through cytokinin oxidase. The results of in-vitro assays carried out with partially purified enzyme from corn kernels substantiale this conclusion. These findings are discussed in relation to recent observations of auxin and cytokinin levels in crown-gall tumours with altered morphology.Abbreviations FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - IP isopentenyladenine - NAA naphthaleneacetic acid - ZR zeatin riboside     

The effect of auxin type and concentration on pecan (Carya illinoinensis) somatic embryo morphology and subsequent conversion into plants

Summary Embryogenic cultures were initiated from immature pecan zygotic embryos. Explants were induced for one week on Woody Plant Medium with either -naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid at 2, 6 or 12 mg/l, then subcultured monthly to fresh basal medium. Observations were made on callus production, embryo formation, and embryo morphology. Somatic embryo morphology and overall callus proliferation were affected by auxin type. Callus proliferation was less extensive and more somatic embryos resembling zygotic embryos were obtained from cultures initiated with -naphthaleneacetic acid than with 2,4-dichlorophenoxyacetic acid. Repetitive somatic embryogenesis was obtained in all auxin treatments. Conversion into plantlets was affected by somatic embryo morphology in that embryos with poorly developed apices exhibited lower percentages of conversion than those with well developed single or multiple apices. Consequently, although more embryos were obtained with 2,4-dichlorophenoxyacetic acid, naphthaleneacetic acid was the superior auxin for production of somatic embryos more likely to convert into plants.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - WPM Woody Plant Medium (Lloyd & McCown 1980)     

Tissue culture and plant propagation of Gomphrena officinalis — a Brazilian medicinal plant

Leaf and stem segments of Gomphrena officinalis originated from aseptically grown seedlings were used to initiate cultures. Callus production was obtained on gelled Murashige & Skoog medium supplemented with 6-benzylaminopurine alone (1.0, 5.0 or 10.0 mgl^-1) or combined with -naphthalene acetic acid (0.1, 0.5 and 1.0 mgl^-1) after 10 to 15 days of culture, and can be transferred to fresh medium every 30 days. The combinations of 5.0 or 10.0 mgl^-1 of 6-benzylaminopurine with 0.1 mgl^-1 of -naphthalene acetic acid were found to be the best for shoot regeneration. Adventitious shoot formation occurred after 50 to 60 days of culture in leaf and internode stem explants. Nodal segments developed actively growing lateral buds after 30 days of culture. Gelled Murashige & Skoog medium containing 10 mgl^-1 of indole-3-butyric acid was considered optimal for the rooting of shoots. Rooted plants transferred to potting soil could be successfully established.Abbreviations BA 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog - NAA -naphthalene acetic acid     

The role of cellulase in hormonal regulation of shoot morphogenesis in tobacco callus

A polyclonal antibody raised against cellulase (EC 3.2.1.4.) from callus ofNicotiana tabacum L. cv. Petit Havana SR1 reduced cellulase activity and induced shoot formation in tobacco callus in the presence of callus maintaining concentrations of auxin and cytokinin. Shoot induction as well as reduction of the cellulase activity was also obtained by withdrawing auxin from the callus medium. The effect of the two hormones on cellulase activity in the tobacco tissue was examined by varying the concentration of one of the hormones -naphthylacetic acid (NAA) or benzylaminopurine (BAP) at a time while the other was kept at a level sufficient for either callus growth or shoot induction. While NAA stimulated the enzyme activity increasingly with concentration in the range 5 × 10^–7 M to 5 × 10^–5 M at both levels of BAP, BAP only stimulated the cellulase activity at an optimum concentration of 5 × 10^–6 M when NAA was present at a level sufficient to induce callus growth. The results point to a pivotal role of the downward regulation of cellulase in the initiation of shoot induction. A series of events leading to oriented cell divisions as a result of the lowered cellulase level during the initial phase of the morphogenetic process is discussed.Abbreviations Ab Purified cellulase antibody - BAP benzylaminopurine - MS Murashige and Skoog medium - NAA -naphthylacetic acid - PS Purified preimmune serum We thank Mr. Poul Fabech for constructing the automatic viscosimetric equipment and Mr. Hans Hjorth for making the computer programme. This work was supported by The Danish Veterinary and Agricultural Research Council.     

Formation of plantlets through somatic embryogeny in the Himalayan blue poppy,Meconopsis simplicifolia (Papaveraceae)

Summary Exuberant and subculturable calli could be induced from only hypocotyl and leaf segments of ca 4-month-old seedlings of Meconopsis simplicifolia cultured on Murashige & Skoog's medium supplemented with 10^–6M kinetin + 10^–5M -naphthalene acetic acid. Suspension cultures were initiated from the calli in a similar medium but with 10^–5M 2,4-dichlorophenoxy acetic acid in place of -naphthalene acetic acid. In ca 80% of the suspension cultures somatic embryos differentiated freely (80–85%) as well as on the surface of small clumps of tissue (15–20%). Somatic embryos that developed beyond heart-shaped stage were transferred to agar-solidified Murashige & Skoog's medium free of growth substances. When maintained in 10 h light and 14 h dark the somatic embryos developed into plantlets bearing cauline leaves. From seed sowing to raising normal plantlets via callus required 28 weeks; on average 80 plantlets were obtained from one explant in three passages.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - Kn kinetin - MS Murashige & Skoog's medium (Murashige and Skoog 1962) - NAA -naphthalene acetic acid     

Analysis of gametophytic development in the moss,Physcomitrella patens,using auxin and cytokinin resistant mutants

Mutants altered in their response to auxins and cytokinins have been isolated in the moss Physcomitrella patens either by screening clones from mutagenized spores for growth on high concentrations of cytokinin or auxin, in which case mutants showing altered sensitivities can be recognized 3–4 weeks later, or by non-selective isolation of morphologically abnormal mutants, some of which are found to have altered sensitivities. Most of the mutants obtained selectively are also morphologically abnormal. The mutants are heterogeneous in their responses to auxin and cytokinin, and the behaviour of some is consistent with their being unable to make auxin, while that of others may be due to their being unable to synthesize cytokinin. Physiological analysis of the mutants has shown that both endogenous auxin and cytokinin are likely to play important and interdependent roles in several steps of gametophytic development. Although their morphological abnormalities lead to sterility, genetic analysis of some of the mutants has been possible by polyethyleneglycol induced protoplast fusion.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - NAA 1-naphthalene acetic acid - 2,4D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - IAP 6-( ²isopentenyl) aminopurine - NAR NAA resistant mutants - BAR BAP resistant mutants     

Propagation of Gasteria croucheri Bak. from shoot producing callus

Leaf explants of Gasteria croucheri Bak. cultured on a modified Murashige and Skoog medium supplemented with 8.8 × 10^-6M (2 mgl^-1) benzyladenine and 2.7 × 10^-5M (5 mgl^-1) -naphthalene acetic acid yielded a callus which produced adventitious shoots. This callus could be maintained on a medium with 1.9 × 10^-5M (4 mgl^-1) kinetin only and continued to produce shoots which could be rooted on a hormone free medium. Plantlets could be readily acclimatized.     

Regeneration in cultures of Papaver bracteatum as influenced by growth hormones and temperature

Callus was induced on Papaver bracteatum Lindl. seedlings inoculated on Murashige & Skoog (1962) medium supplemented with -naphthaleneacetic acid (NAA) (1.0 mg 1^-1) and benzylamine purine (BA) (0.5 mg 1^-1). Subculture resulted in excellent callus proliferation but no organogenesis. Shoots were regenerated in cultures grown on MS medium containing NAA (1.0 mg 1^–1), BA (0.5 mg 1^–1) and casein hyrdrolyzate (2.0 mg 1^-1). The shoots developed into plantlets after 8 weeks of culture, and were induced to root on 1/2 MS without the addition of growth regulators. The rooted plantlets were transferred to soil after hardening.MS at full strength was found inhibitory for callus induction and proliferation, but 1/2 MS was suitable. Similarly callus growth was very slow at 25°C but increased when the temperature was lowered to 15°C as did bud initiation.Abbreviations BA benzylamine purine - IAA indoleacetic acid - K kinetin (6-furfurylamino purine) - NAA -naphthalene acetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid     

Cytokinin metabolism in nondividing and auxin-induced dividing explants ofHelianthus tuberosus L. Tuber tissue

Aqueous solutions of auxin (indole-3-acetic acid,-naphthalene acetic acid, or 2,4-dichlorophenoxyacetic acid) were active in inducing DNA synthesis and mitosis in prewashed tissue explants of mature Jerusalem artichoke tubers. Explants did not respond in this way to aqueous solutions of cytokinin (zeatin, zeatin riboside, 6-benzylaminopurine, or kinetin). The metabolism of [8-³H]zeatin riboside (ZR) was studied in non-dividing and auxin-induced synchronously dividing explants over the first 36 h of culture. ZR was taken up rapidly and to the same extent by both tissues. Sequential analysis of tissue extracts by thin-layer and high-performance-liquid chromatography identified zeatin nucleotide(s) (ZN), O-glucosyl zeatin riboside (OGZR), adenosine, and adenine nucleotide(s) (AN) as the principal metabolites in both tissues. The proportion of radio-activity due to ZR declined steadily and OGZR accumulated steadily at similar rates in both tissues. ZN was the major metabolite in both tissues at 12 h; thereafter ZN continued to accumulate in nondividing tissue, but its level declined in dividing tissue, and a corresponding increase in the levels of AN and adenosine was observed. These treatment differences in cytokinin metabolism were apparent at least 6 h before the onset of mitosis.     

Influence of various media constituents on the growth of Cinchona ledgeriana tissue cultures and the production of alkaloids and anthraquinones therein

Using the methods reported by De Fossard et al. (11) the influence of various media constituents on the growth and the alkaloid and anthraquinone production in Cinchona ledgeriana callus cultures was studied. Growth and indole alkaloid production (e.g. cinchonamine) was improved by higher auxin levels. The best growth was observed in the light, although many media resulted in no growth at all in the light. Anthraquinone production was highest at lower auxin levels. Quinoline alkaloid levels (e.g. quinidine) were highest in media with low auxin concentrations. Low and medium cytokinin concentration benefited the quinoline alkaloid production.From the results it was concluded that the pathways leading to the various secondary products, anthraquinones, indole alkaloids and quinoline alkaloids are, at least partly, regulated independently.Abbreviations used IAA indol-acetic acid - IBA indol-butyric acid - NAA -naphtaleneacetic acid - NOA 2-naphtoxy-acetic acid - 2,4-D 2,4-dichlorophenoxy-acetic acid - pCPA parachlorophenoxy-acetic acid - BA benzyladenine     

Methyl jasmonate and bean leaf abscission

The effect of rac-methyl jasmonate, both in solution and as a vapour, on the separation of pulvinar and petiolar tissues in explants containing the distal abscission zone of primary leaves of Phaseolus vulgaris var. Contender was investigated. The effects of rac-methyl jasmonate were compared to those of (±)-abscisic acid, -naphthalene acetic acid, ethylene and 2-chloroethylphosphonic acid. Abscission times were determined in explants prepared from 14-day-old control plants and in explants prepared from plants that had been pretreated for 24h with the ethylene-action inhibitor, silver thiosulphate. While silver-pretreatment, or treatment with -naphthalene acetic acid delayed abscission, treatment with ethylene or 2-chloroethylphosphonic acid accelerated tissue separation. However, (±)-abscisic acid delayed abscission under these conditions. In all instances, treatment with rac-methyl jasmonate had no apparent effect on abscission. The loss of chlorophyll from bean leaf discs incubated in the dark was enhanced by treatment with 2-chloroethylphosphonic acid or (±)-abscisic acid and was retarded in discs incubated in benzyl adenine. While incubation in -naphthalene acetic acid was without effect, incubation in solution of rac-methyl jasmonate also retarded chlorophyll loss when compared to water controls.     

Regeneration of Lycium barbarum L. plants from leaf tissue,callus culture and callus protoplasts

The possibility of plant regeneration from leaf tissue, callus and callus protoplasts of Lycium barbarum L. has been studied. Leaf segments were cultured on B5 medium (Gamborg et al. 1968) containing 1.5 mg/1 6-benzylaminopurine and 0.5 mg/1 -naphthaleneacetic acid. Regeneration of shoots was initiated after 30 days of cultivation. Callus was obtained from leaf and internode tissues on MS medium (Murashige and Skoog 1962) containing 0.4 mg/1 of 2,4dichlorophenoxyacetic acid. Subsequently, callus was successfully subcultured on the same medium with 1 mg/l of 2,4-dichlorophenoxyacetic acid and 0.2 mg/l -naphthaleneacetic acid. Organogenesis in callus culture was obtained in the course of 40 days after transferring to TM-4 (Shahin 1984). Protoplasts were isolated from callus tissue grown in vitro using an enzymatic method. Cell colonies, minicallus formation and organogenesis were obtained. Shoots were rooted on Murashige and Skoog medium containing 0..1 mg/l -naphthaleneacetic acid. Regenerated plants were transferred to soil and were grown to maturity. Regenerated plants carried normal morphological traits.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - Zea zeatin - GA₃ gibberellic acid - MS Murashige and Skoog medium - B5 Gamborg medium     

Correlation between the presence of membrane-bound auxin binding and root regeneration in cultured tobacco cells

When cell-suspension cultures and callus tissue from Nicotiana tabacum are grown on medium containing -naphthaleneacetic acid (NAA) and kinetin, three classes of auxin-binding proteins can be detected. When the herbicide 2,4-dichlorophenoxyacetic acid is used instead of both NAA and kinetin, one of these sites, which is membranebound, disappears. After retransferring cells to medium containing NAA and kinetin, this membrane-bound site reappears after four to eight weeks. This reappearance is correlated with the ability of the cells to regenerate roots.Abbreviations IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Changes in phenylalanine ammonia-lyase activity during xylem differentiation in Coleus and soybean

Summary Xylem differentiation was induced in cultured Coleus internode slices when grown in the light on a simple agar/sucrose/IAA medium and in darkgrown soybean callus tissue when cultured on a complex defined medium containing 5×10^-7 M kinetin. In the Coleus system, the activity of phenylalanine ammonialyase followed the same time course as the formation of lignified wound vessel members. The specific activity of PAL was higher in the soybean callus tissues grown on 5×10^-7 M kinetin, which produced tracheary elements, than in the soybean tissue grown on 10^-8 M kinetin, which did not produce tracheids. These observations suggest that PAL is a marker enzyme for xylogenesis and that PAL activity may be a rate limiting step in lignification.Abbreviations IAA indole 3-yl acetic acid - NAA -naphthalene acetic acid - 2,4D 2,4,dichlorophenoxyacetic acid - DNA deoxyribose nucleic acid - TCA trichloracetic acid - PAL phenylalanine ammonia-lyase