Combinations Of Cytokinins Research Articles

Plant Regeneration from Pea Leaflets Cultured in vitro and Genetic Stability of Regenerants

Bud and shoot formation and regeneration of complete plants have been achieved with immature and mature leaflets, at various frequency, under the influence of a range of cytokinin and auxin combinations. Immature leaflets, 0.9 to 1.8 mm in length, produced shoots in 26 to 38% explants on a medium containing 10.0 μM BA and NAA, IBA or IAA. A reduction in the frequency of bud and shoot regeneration to 7% in the presence of 10.0 μM each of BA + NAA was observed with mature leaflets. In the differentiation of buds and shoots, no stimulating effect of GA(3) has been observed. Combinations of cytokinins with picloram and 2,4-D suppressed organogenesis. Plant regeneration was influenced by genotype and temperatures. Seed-derived progeny of regenerants contained the normal diploid number of chromosomes (2n = 14). Analysis of four isozymes, esterase, GDH, 6-PGD, and LAP, as well as nine genetically defined morphological characters indicated retention of genetic stability in the progeny of tisssue culture propagules.

The Effects of Glyphosate, Auxin, and Cytokinin Combinations on in Vitro Development of Cranberry Node Explants

Abstract Axillary shoot proliferation from cranberry (Vaccinium macrocarpon Ait.) nodes in vitro was correlated positively with glyphosate [(N-(phosphonomethyl) glycine,(Gly)] and isopentenyladenine (2iP) concentrations. Maximum proliferation was caused by 3.7 × 10−3 m Gly or 1.5 × 10−4 m 2iP. Shoot proliferation was reduced when combined at levels above 1.9 × 10−3 m Gly and 7.4 × 10−5 m 2iP. Gly and 2iP reduced shoot and root dry weight. Indoleacetic acid (IAA) did not significantly affect shoot proliferation or shoot and root growth at concentrations of 5.7 × 10−6 m and 5.7 × 10−5 m. Gly induced basal swelling of shoots and reduced leaf size. These characteristic Gly effects were not altered by 2iP or IAA. Gly may be a useful addendum for in vitro shoot proliferation and a tool for the investigation of meristem initiation and development.

Tissue Culture of Cannabis sativa L. and in vitro Biotransformation of Phenolics

Summary The production of secondary metabolites and the biotransformation of precursor compounds by plant tissue cultures may lead to both the commercial synthesis of valuable substances and an elucidation of their biosynthetic pathways. Callus and suspension cultures of Cannabis sativa L. were induced from embryo, leaf and stem expiants with various combinations of auxins and cytokinins. The optimal ratio of plant growth regulators for callus induction and subsequent cell proliferation differed according to the source of the expiant and the mode of culture. Cannabis suspensions, inoculated with cannabidiol, produced two major cannabinoids as assayed by thin-layer-chromatography. Gas chromatographic/mass spectral analysis identified both phenolics as diastereoisomers of cannabielsoin. Suspension cultures inoculated with olivetol produced an unidentified cannabinoid with a molecular ion of m/z 210.

Field Evaluations of Morphactins and Other Growth Regulators for Senescence Delay of Flax, Soybean, Wheat, and Oats1

Field trials were conducted from 1975 through 1979 to detect chemicals which would delay senescence of flax (Linum usitatissimum L.), soybean [Glycine max (L.) Merrill], wheat [Triticum aestivum (L.) em Thell], and oats (Avena sativa L.). Of 18 regulators tested 2 or more years on flax, 11 delayed leaf yellowing after single applications in at least I year. The morphactin chlorflurenol (methyl 2‐chloro‐9‐hydroxyfluorene‐9‐carboxylate), applied postbloom at rates of 25 to 50 g/ha retarded losses of chlorophyll and leaf area and promoted shoot growth and flowering. Early‐flowering cultivars were more responsive than late cultivars. Dichlorflurenol (methyl 9,7‐dichloro‐9‐hydroxyfluorene‐9‐carboxylate) and n‐butyl flurenol (n‐butyl 9‐hydroxyfluorene‐9‐carboxylate), were slightly less and markedly less effective than chlorflurenol, respectively. Other regulators which were effective when applied postbloom were 3‐CPA (2‐(3‐chlorophenoxy)propionamide), TIBA (2,3,5‐triiodobenzoic acid), ethephon (2‐chloroethyl)phosphonic acid), SD‐8339 (N‐ (phenylmethyl)‐9‐(tetrahydro‐2H‐pyran‐2‐yl)‐9‐ H‐purin‐6‐amine), BA + GA4,7(6‐(benzylamino) purlne plus gibberellins 4 and 7), and PP528 (ethyl 5‐(4‐chloro‐phenyl)‐2H‐tetrazole‐2‐acetate). Treatments made earlier than postbloom often resulted in growth abnormalities. Combination of cytokinin or gibberellin with other regulators usually did not enhance effectiveness.Chlorflurenol delayed yellowing of ‘Swift’ soybean when applied in R5 stage at rates of 25 and 50 g/ha. Applications at higher rates or earlier dates caused growth abnormalities. Other effective chemicals included dichlorflurenol, BA, and BA + GA4,7. For oats and wheat, chlorflurenol was ineffective when applied from boot stage to postanthesis at rates up to 1000 and 2000 g/ha, respectively.

In vitro fertilization and seed development inTrifolium

Pistils ofTrifolium repens L. andT. ambiguum Bieb. were cultured on an agar-based modified Murashige-Skoog medium. Pistils with and without accessory floral parts were removed from flowers of selected clones ofT. repens, hand-pollinated under aseptic conditions, and planted on the medium. Pistils cultured without accessory floral parts showed no evidence of fertilization after 2 weeks. However, 52% of thoseT. repens pistils cultured with calyx lobes and pedicels contained ovules with maturing embryos 12 days after in vitro cross-pollination. Pistils fromT. ambiguum intraspecific cross-pollinations could not be cultured successfully under the same conditions; however, addition of various combinations of auxin, cytokinin, and gibberellic acid enhanced embryo growth. Fertilization and partial embryo development occurred in interspecific crosses betweenT. ambiguum andT. repens orT. hybridum only whenT. ambiguum was used as the pistillate parent. These results indicate that embryological development under in vitro conditions closely parallels in situ development although growth regulator requirements may vary among species.

Shoot Development in Established Callus Cultures of Cultivated Tomato (Lycopersicon esculentumMill.)

Summary Four established callus lines of Lycopersicon esculentum Mill . and one of L. pimpinellifolium Mill ., ranging in age between 17 and 28 months, were grown on media containing various combinations of cytokinins (Z, BA, and 2ip) and IAA in an effort to induce shoot development. With all 3 cytokinins shoots formed only in one genotype, VFNT Cherry, and only after several months of regular transfers to fresh medium. When callus from plated suspension cultures derived from the 5 callus lines was grown on medium containing Z and IAA, shoots developed only from the VFNT Cherry callus. Four 4-month old callus lines of L. esculentum were also tested and VFNT Cherry was still the only genotype to produce shoots. The shoots produced by the 4-month old VFNT Cherry callus were normal in appearance and rooted readily. All shoots produced by the older cultures were morphologically abnormal and could not be induced to root.

Multiple-drop-array (MDA) technique for the large-scale testing of culture media variations in hanging microdrop cultures of single cell systems. II. Determination of phytohormone combinations for optimal division response in Nicotiana tabacum protoplast cultures

The effects on cell division of 7 auxin-like growth substances and 4 cytokinins were examined in Nicotiana tabacum protoplast cultures by the use of the multiple-drop-array (MDA) technique. The phytohormones were tested in 1 auxin/1 cytokinin combinations over a wide range of concentrations. Conditions were found which resulted in routine division frequencies of 90–95% for mesophyll protoplasts of wild-type and two light-sensitive mutants of tobacco.

In vitro Plantlet formation from hypocotyl and hypocotyl callus of Cucumis sativus L. cv. 'Shinko Fushinari No.10

The purpose of this study was to grope the hormonal requirements for the differentiation of complete plantlet from hypocotyl and hypocotyl callus of Cucumis sativus L.cv. 'Shinko Fushinari No.10'. At first, segments of hypocotyl (1 cm) in young seedling were inoculated on the agar media (MURASHIGE & SKooG's basal medium) with various combinations of auxin (NAA) and cytokinin (BA) concentration. Subsequently, half of the cultures were untouchedly cultured on the same media, not transferring hypocotyl callus to another medium. The callus formed from other half hypocotyl were transferred to another media, that is two kinds of media with 1.0ppm BA alone and in the absence of NAA and BA, being di-vided into the small mass the size of an adzuki bean. Callus formation from hypocotyl required alternative or both addition of auxin and cytokinin at O.1ppm or higher levels of concentration, although callus was favourably fcrmed for the most part. Root formation was observed at the media with the combination of O-10.0ppm NAA and O-1.0ppm BA and the ratio of root formation generally increased as BA concentration becomed lower. On the other hand, shoot formation was observed at a low frequency on the media with BA concentration either higher than or equal with NAA concentration, that is, with the combination of O-1.0ppm NAA and 1.0ppm BA. However, only a few of the shoots continued to develop and one complete plantlet was finally obtained at the medium with O.O1ppm NAA and 1.Oppm BA. Shoot formation from the transferred callus was observed at a low frequency only when calluses produced on the medla wlth the combinatron of O.O1-1Oppm NAA and 1.Oppm BA were transferred to the two kinds of media for redifferentiation. It appeared from this result, as lettuce and tomato, that whether shoot was formed from callus or not was destined by the relative concentration between auxin and cytokinin in the medium for callus induction. Complete plantlets were obtained at a low frequency on the meium in the absence of NAA and BA. Root formation was Observed only when shoot vv-as L0rmed on the media in the absence of NAA and BA.

In vitro culture of cotyledon tissue of Castanea sativa Mill

Establishment of callus tissue culture from cotyledon fragments of Castanea sativa Mill. is described. The explants were grown on various combinations of auxins (IAA, IBA, NAA, 2,4-D) and cytokinins (K, BA) to induce callus and regeneration of organs. The best growth occurred on basal medium with 2,4-D (1 and 10 mg/l) plus both K or BA (0.5 mg/l). Root regeneration was achieved with both IBA and NAA (10 mg/l) supplemented with K or BA.

Response of Phony-infected Peach Trees to Gibberellic Acid1

Abstract Peach trees (Prunus persica (L.) Batsch) dwarfed by phony disease responded to 2 summer applications of 264 ppm gibberellic acid (GA) by breaking the disease-induced rest period and resuming nearly normal twig growth. No twig growth was produced on untreated trees and no significant growth occurred on phony trees treated with combinations of cytokinins, sodium isoascorbate, calcium nitrate, urea, or citric acid.

Defined conditions for the initiation and growth of cotton callus in vitro. I. Gossypium arboreum.

Defined in vitro conditions for callus initiation by Gossypium arboreum L. were determined, and different tissues were evaluated as explant sources, Environmental conditions tested included light versus dark, and low light versus high light. Different nutrient media as well as carbohydrate sources were examined. Our data show that hypocotyl tissue was superior to cotyledon or leaf tissue as the explant source for callus proliferation; the Murashige-Skoog inorganic formulation with (in mg per 1) 100 myo-inositol, 0.4 thiamine-HCl, 2 indoleacetic acid (IAA), 1 kinetin, and 3% glucose solidifield by agar was the best medium to initiate callus. Cultures with sucrose as a carbohydrate source browned rapidly. Callus proliferation was superior under high light (8000 to 9000 lux) conditions at 20 +/- 1 degree C. Various combinations of auxins and cytokinins were tested for their ability to improve callus proliferation and subsequent growth of subcultures. Although the MS medium containing IAA and kinetin was found superior for obtaining rapid proliferation of callus from hypocotyl explants, a second medium containing 2 mg per 1 naphthaleneacetic acid (NAA) and 0.5 to 1 mg per 1 benzyladenine (BA) was found necessary for vigorous growth of subcultured callus. A MS medium with 5 to 10 mg per 1 N6-[delta2-isopentenyl]-adenine (2ip) and 1 mg per 1 NAA was also favorable for continued subculturing.

Altering the Sensitivity of Carnation Flowers (Dianthus caryophyllus L.) to Ethylene1

Abstract Frequent irrigation regimes, and cool storage increased the sensitivity of carnation cut flowers to ethylene which was reflected by a shorter residual flower life. A combination of cytokinins, 0.23 m<sc>M</sc> kinetin or isopentyl adenine (IPA), with 5% w/v sucrose reduced the sensitivity to ethylene and increased longevity 2 to 2.6 times, respectively. The reversible and irreversible wilting response to ethylene is discussed.

Plant Differentiation in Callus Tissue Induced from Immature Endosperm of Oryza sauva L.

Immature endosperms of the rice plant ( Oryza sauva L.) harvested 3–7 days after pollination were implanted into various culture medium. Callus induction was observed in cellular endosperm containing starch grains 4–5, 5–6 and 6–7 days after pollination. On the other hand, it wasn't observed in coenocytic endosperm containing no starch grains 3–4 days after pollination. Callus tissues induced from immature endosperms in the various culture medium were subcultured in modified White medium + 0.4% yeast extract + 10 -5 M 2,4-D. The effect of various concentration combinations of auxin and cytokinin was tested on organ formation. Roots differentiation was often observed in subcultured endosperm callus. Two plantlets were also obtained and totipotency of endosperm was confirmed.

Effects of growth regulators on fatty acids of soybean suspension cultures

Soybean suspension cultures were grown for 24 weeks in the absence of plant growth regulators and in the presence of 1 ppm levels each of an auxin (indole-3-butyric acid), a cytokinin (kinetin) and a gibberellin (gibberellic acid), individually and in all possible combinations. Cells grown in the presence of the auxin with and without gibberellin contained relatively greater amounts of palmitic and smaller amounts of polyunsaturated acids than did cells grown under other regimens. The combination of cytokinin and gibberellin caused a higher proportion of linoleic and a lower proportion of linolenic acids than in cells of the other groups. Neither of these regulators by itself produced the effect, and addition of auxin to the other two diminished the effect.

Hormonal Control of Differentiation of Shoots, Roots and Embryos in Leaf and Stem Cultures of Petunia inflata and Petunia hybrida

AbstractFactors influencing adventitious bud and root development, callus induction and embryogenesis were investigated in stem and leaf cultures of Petunia inflata R. E. Fries and Petunia hybrida cv. Cascade and cv. Rose du ciel grown on a synthetic nutrient medium. Indoleacetic acid caused limited callus development and root formation whereas naphthaleneacetic acid Induced abundant roots. 2,4‐Dichlorophenoxyacetic acid promoted callus growth and differentiation of embryos which eventually developed into plantlets. Cytokinins such as benzyladenine, zeatin and kinetin induced bud development. A combination of auxins and cytokinins caused an interaction which was manifested in altered morphogenetic response. Thus 2,4‐dichlorophenoxyacetic acid in conjunction with benzyladenine caused suppression of bud development and retarded differentiation of embryos. Likewise, when benzyladenine was used with indoleacetic acid root development was totally inhibited and abundant buds were produced.

<i>In vitro</i> regeneration of pumpkin (<i>cucurbita maxima</i>) through shoot apical meristem

Context: Pumpkin (Cucurbita maxima) is very important vegetable crop. It is damaged by several types of diseases, especially viral diseases which reduce the total yield of pumpkin. This is very difficult problem and such type of problem can be overcome by meristem culture. Objectives: To develop suitable protocols for indirect regeneration of pumpkin plant through shoot apical meristem. Materials and Methods: Meristems were isolated from 15-21 days in vivo grown plants by collecting shoot tips and were prepared (0.2-0.5 mm) under 4X zoom stereo-microscope remaining two leaves primordia. Inoculation of explants was made singly per culture vessel in semisolid MS fortified with different concentrations and combinations of cytokinin and auxin. Results: Among different concentrations, 1.0 mg/l BAP showed best response for callus induction. Calli were sub cultured for shoot formation in MS containing BAP singly or combination with GA3 and 1.5 mg/l BAP + 0.1 mg/l GA3 gave better result. In vitro regenerated shoots were rooted well in ½ strength MS with 0.5 mg/l IBA and micro plants were acclimatized successfully in natural condition. Conclusion: Indirect regeneration of pumpkin plants through shoot apical meristem has been established. Keywords: Growth regulators; in vitro regeneration; Pumpkin; Cucurbita maxima. DOI: http://dx.doi.org/10.3329/jbs.v18i0.8784 JBS 2010; 18(0): 104-107

High Frequency <i>In Vitro</i> Propagation of <i>Adhatoda vasica</i> Nees through Shoot Tip and Nodal Explants Culture

An efficient protocol for in vitro propagation of Adhatoda vasica Nees was established using shoot tip and nodal explants from field grown mature plant. Proliferation of multiple shoots was achieved on MS medium supplemented with different concentrations and combinations of cytokinins (0.5-4.0 mg/l) and auxins (0.1-1.0 mg/l). Maximum number of shoots per explant (13.0) was obtained on MS medium supplemented with 2.0 mg/l BAP + 0.2 mg/l NAA. Among two types of explants used in this study, nodal explants showed better response in respect of multiple shoot production. The elongated shoots were excised and subcultured for rooting on MS medium supplemented with different concentrations of auxins (IBA and NAA). Highest 80% rooting was achieved; and three to four roots per shoot were recorded in medium with 1.0 mg/l IBA within 4 weeks of culture. The in vitro raised plantlets were acclimatized and successfully transferred to natural condition in pot. The regenerated plants were healthy, uniform and identical to the donor plants and the survival percentage was 80%. Key words: Micropropagation, Adhatoda vasica, shoot tip, nodal explant. DOI:10.3329/jbs.v16i0.3739 J. bio-sci. 16: 35-39, 2008

<i>In Vitro</i> Micropropagation of Orchid (<i>Vanda tessellata</i> L.) from Shoot Tip Explant

Context: Orchid produces a huge number of minute seeds but the seeds can not germinate easily in nature due to the lack of endosperm in the seeds is an incompatibility barrier that limits its propagation in nature. Objectives: To develop in vitro culture techniques for quick propagation of Vanda tessellate, a commercially important orchid species. Materials and Methods: Shoot tips were used as experimental materials. The explants were surface sterilized and the shoot tips were excised. The isolated shoot tips were cultured in MS medium supplemented with different concentration and combinations of auxin and cytokinin. Results: The combination of 1.5 mgl-1 NAA and 1.0 mgl-1 BAP was proved to be the best medium formulation for multiple shoot formation as well as maximum shoot elongation. The single shoots were isolated from the multiple shoots and subcultured in MS medium having NAA and IBA individually and in combinations for root induction. Maximum root induction was obtained in MS agarified medium having 0.5 mgl-1NAA and 1.0 mgl-1IBA. The well rooted plantlets were hardened successfully in the potting mixture containing coconut husk, perlite, charcoal, brick pieces in the ratio of 2:1:1:1 and eventually established under natural condition.Conclusion: An efficient regeneration protocol for micropropagation in V. tessellata through shoot tip culture has been established.Key words: Shoot tip; micropropagation; orchid.DOI: 10.3329/jbs.v17i0.7122J. bio-sci. 17: 139-144, 2009