Seed potato (Solanum tuberosum L.) yield and tuber number increase after foliar applications of cytokinins and gibberellic acid under field and glasshouse conditions

The purpose of these experiments is to determine the effects of foliar applications of benzylaminopurine (BAP) and gibberellic acid (GA) on tuber number production of seed potatoes. In field experiments conducted during 1989/90 cv. Mailén was used and BAP, 50 mg·l^–1 was foliarly applied at (1) tuber initiation, 36 days after emergence (DAE); (2) 54 DAE; and, (3) 64 DAE. Under glasshouse conditions, in 1991/92 cv. Spunta was used and BAP 50 mg·l^–1+GA 50 mg·l^–1 were applied 30 and 37 days after planting/transplanting. In 1992 cv. Huinkul, Kennebec and Spunta were used and BAP 50 mg·l^–1+GA 50 mg·l^–1 and Biozyme (Techic SA), a commercial product with auxin (IAA, 32.2 mg·l^–1), gibberellic acid (GA₃, 32.2 mg·l^–1) and cytokinins (zeatin, 83.2 mg·l^–1) at 5 ml·l^–1 were applied. In cv. Mailén, a higher tuber number in the seed fraction (<80 g) was found when BAP was applied at each of the three crop stages, while applications 54 and 62 DAE also increased tuber number in the 80–400 g fraction. As a result of BAP applications, tuber yield was also significantly increased. In the glasshouse experiments, cv. Spunta showed a significant increase in minituber production in 3 out of 4 cases, either if the mother plant came from in vitro generated plantlets or minitubers, or if GA + BAP or Biozyme was applied. It can be concluded that the use of these PGRs under both field and glasshouse conditions in cvs. Mailén and Spunta can result in increased tuber number in the seed fraction.     

Effects of kinetin,paclobutrazol and their interactions on the microtuberization of potato stem segments cultured in vitro in the light

Single-nodal cuttings of Solanum tuberosum (four cultivars) and Solanum chacoense were induced to produce in vitro microtubers on Murashige & Skoog (MS) medium supplemented with 8 g l^–1 sucrose and various concentrations of kinetin and paclobutrazol. The cultures were kept 10 days in darkness and then transferred to a 14 h daylength with 100 µE m^–2 sec^–1 light intensity at 21 °C. Kinetin (2.5 mg l^–1) had no significant influence on tuber formation. However, its addition together with paclobutrazol (0.001 mg l^–1) significantly enhanced tuberization. Paclobutrazol alone stimulated early tuber initiation and inhibited stem growth. Despite some genotype × treatment interactions, all genotypes (from very early to late and wild type) formed the maximum proportion of explants bearing microtubers on the media containing both plant growth regulators.     

Axillary shoot proliferation and tuberization of <Emphasis Type="Italic">Dioscorea fordii</Emphasis> Prain et Burk

Dioscorea fordii Prain et Burk., a member of the family Dioscoreaceae, is an important tuberous food in China owing to its edible and medicinal functions. However, the lack of healthy planting material has restricted its production. The present study was an attempt to develop a protocol for in vitro propagation of D. fordii. In an initial two by two (2²) factorial experiment, the effects of culture system and activated charcoal have been observed for axillary shoot proliferation and tuberization of in vitro plantlets. Shoot length, frequency of proliferation, fresh weight and dry weight of shoots, frequency of tuberization and mean number of tubers per plantlet (NTPs) were significantly (P ≤ 0.05) greater in liquid medium compared to semi-solid media. Secondly, an orthogonal experimental design [L₉ (3⁴)] was used to investigate the effects of the ratio of naphthalene acetic acid (NAA) to 6-benzyladenine (BA) (NAA/BA), paclobutrazol, methyl jasmonate and sucrose concentration on tuberization. Sucrose concentration had the record effect on frequency of tuberization, NTPs and frequency of proliferation. The preferred medium for axillary shoot proliferation and tuberization of D. fordii found to be MS basal medium (Murashige and Skoog in Physiol Plant 15:473–479, 1962) supplemented with 1.0 mg l⁻¹ BA, 0.1 mg l⁻¹ NAA, 30 g l⁻¹ sucrose and 1.5 g l⁻¹ AC in liquid culture.     

Direct Shoot Bud Formation and Tuberization from Aseptically Cultured Root Tubers of Calla Lily (Zantedeschia aethiopica L)

We describe here the development of a micropropagation protocol for mass multiplication of Zantedeschia aethiopica by using root tubers as explant. The surface sterilized root tubers produced five to six shoot-buds on semi-solid Murashige and Skoog’s (MS) medium with 10.0 mg l^?1 of 6-benzylaminopurine (BAP) and additives (50.0 mg l^?1 of ascorbic acid; 25.0 mg l^?1 each of adenine sulphate, L-arginine and citric acid). The cultures were multiplied by sub-culture of individual shoot bud produced in vitro and clumps of shoot buds generated in vitro in cultures on MS medium containing 3.0 mg l^?1 of BAP and additives. Further multiplication of propagules was achieved through tuber formation along with amplifying shoots on MS medium with 5.0 mg l^?1 of BAP. The micropropagated shoots were rooted both in vitro as well as ex vitro. Cent percent of the cloned shoots rooted in vitro within 15–18 days on hormone-free 1/2 strength MS salts with 200.0 mg l^?1 of activated charcoal. Alternatively 95–100% shoots rooted ex vitro under greenhouse conditions on soilrite after pulse-treatment with 500.0 mg l^?1 of Indole-3-butyric acid (IBA) or β-naphthoxyacetic acid (NOA) for 300 sec. The cloned plants were hardened in the greenhouse. The hardened plants were transplanted to soil for further acclimatization.     

The Role of Gibberellin, Abscisic Acid, and Sucrose in the Regulation of Potato Tuber Formation in Vitro

The effects of plant hormones and sucrose (Suc) on potato (Solanum tuberosum L.) tuberization were studied using in vitro cultured single-node cuttings. Tuber-inducing (high Suc) and -noninducing (low Suc or high Suc plus gibberellin [GA]) media were tested. Tuberization frequencies, tuber widths, and stolon lengths were measured during successive stages of development. Endogenous GAs and abscisic acid (ABA) were identified and quantified by high-performance liquid chromatography and gas chromatography-mass spectrometry. Exogenous GA_4/7 promoted stolon elongation and inhibited tuber formation, whereas exogenous ABA stimulated tuberization and reduced stolon length. Indoleacetic acid-containing media severely inhibited elongation of stolons and smaller sessile tubers were formed. Exogenous cytokinins did not affect stolon elongation and tuber formation. Endogenous GA₁ level was high during stolon elongation and decreased when stolon tips started to swell under inducing conditions, whereas it remained high under noninducing conditions. GA₁ levels were negatively correlated with Suc concentration in the medium. We conclude that GA₁ is likely to be the active GA during tuber formation. Endogenous ABA levels decreased during stolon and tuber development, and ABA levels were similar under inducing and noninducing conditions. Our results indicate that GA is a dominant regulator in tuber formation: ABA stimulates tuberization by counteracting GA, and Suc regulates tuber formation by influencing GA levels.     

In vitro induction of axillary potato microtubers and improvement of their quality

Germinated tubers of selected cultivars Kera, Resy, Nicola and Oreb were made healthier by heat treatment. They were derived from germ explants on MS media with the addition of BAP 1 mg 1^-1 and IAA 0.2 mg 1⁻¹. After sufficient multiplication of stems, optimum conditions of the photoperiod were followed for the induction of axillary microtubers on stem segments in media with BAP 10 mg 1^-1 and 8% sucrose. The ability of tuberization is different: the early cvs. Kera and Resy induce earlier tubers at a long photoperiod and late cvs. Nicola and Oreb tuber rather at a short photoperiod. In suitable photoperiods the inhibitory substances accelerate the induction of axillary tubers and limit the formation of adventitious roots. Synthetic inhibitors applied in induction media increase the number of the tubers. The quality of tubers was affected by the addition of a mixture of amino acids: aspartic, glutamic, lysine and proline in concentrations of 12.5, 25 and 50 mg.1^-1 into the induction media. In the course of cultivation the types which were growing well, formed tubers and increased the volume were selected. The representation of amino acids in the tubers was not significantly affected, there was only an increase in proline. The higher content of amino acids was reflected in the increase of proteins in the tubers. The selected clones are further multiplied.     

High K<Superscript>+</Superscript> does not affect potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) tuber induction,but represses its development <Emphasis Type="Italic">in vitro</Emphasis>

The role of K⁺ in potato (Solanum tuberosum L.) tuberization, based on the effects of K fertilizer and soil exchangeable K⁺, appears to be mostly contradictory. Here, we provide evidence that K⁺ at high concentrations is detrimental to tuber development in vitro once induction has taken place. An experimental system using in vitro-cultured single-node cuttings showed that K⁺ at ≥30.0 mM significantly reduced tuber fresh mass concomitant with a corresponding decline in starch content. However, high K⁺ did not affect tuber induction in terms of number of tubers developed per cutting. High K⁺-induced inhibitory effect on tuber development was attributed to a reduced rate of assimilate partitioning. ⁸⁶Rb(K) transport to stolons, and tubers that acted as strong sinks in vitro were proportional to exogenous K⁺ levels; however, ⁸⁶Rb accumulation and K⁺ deposition were markedly reduced in tubers as compared with that in stolons, especially at higher K⁺ levels. The results indicated a diminishing sink strength developed by tubers with increasing K nutrition. Highly significant negative correlations between ⁸⁶Rb accumulation/K⁺ deposition in both the sink organs and tuber fresh mass reinforced the inhibitory effect of high K⁺ on tuber development. The rate of tuber K removal in vitro was similar to that of crop K removal reported in vivo, suggesting highly conserved K uptake and transport mechanisms during tuberization process. The results have been discussed in the context of possible effects of high K⁺ on impairing sucrose uptake and metabolism.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Chimonanthus praecox</Emphasis> (L.), a winter flowering ornamental shrub

A procedure for the micropropagation of Chimonanthus praecox (L) Link, wintersweet, has been developed using buds from adult trees excised in spring. Shoot cultures established on Murashige and Skoog (1962) medium supplemented with 0.5 mg l⁻¹ 6-benzyladenine (BAP) and 0.1 mg l⁻¹ indole-3-butyric acid (IBA) were difficult to maintain in vitro through extended periods of time due to browning of the medium, shoot and leaf necrosis, and hyperhydricity. A treatment combining the use of 0.1% w/v activated charcoal and addition of a double phase agar-solidified/liquid medium improved propagation, enabling a successful in vitro propagation scheme to be developed. Optimal shoot multiplication occurred on medium containing 0.5 mg l⁻¹ BAP, and rooting on medium with 2.0 mg l⁻¹ IBA for 7 d, followed by transfer to hormone-free medium. Rooted plantlets were easily acclimated in a glasshouse and replanted and cultured outdoors.     

Clonal propagation of Enset (Ensete superbum (Roxb.) Cheesman) through shoot tip cultures

Summary The morphogenetic potential of the shoot tip explants ofEnsete superbum (Roxb.) Cheesman, a wild relative of the cultivated bananas, was investigated and an effective clonal propagation method devised. Shoot tip explants grown in modified MS medium containing 1.5 mg l^–1 BAP and 1 mg l^–1 KIN developed corms which on transfer to medium containing 3 mg l^–1 IBA and 1.5 mg l^–1 BAP, regenerated a large number of shoots from the surface of the corm, the origin of which was traced to single hypodermal cells. Shoots were rooted on a half-strength MS medium salts containing 3 mg l^–1 IBA and 0.1 mg l^–1 BAP. The rooted plantlets were hardened and planted in the field where the plants looked normal.     

Somatic embryogenesis and plant regeneration in centipedegrass (Eremochloa ophiuroides [Munro] Hack.)

Centipedegrass (Eremochloa ophiuroides [Munro] Hack.) is an important warm-season turfgrass and pasture grass. To explore the potential use of biotechnical tools in breeding of centipedegrass, we established an efficient plant regeneration system for this species. Four basal media and 24 combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BAP) were examined for their effects on callus induction from mature seed explants. Twenty combinations of naphthaleneacetic acid (NAA) and BAP were tested for their effect on plant regeneration. Results indicated that Murashige and Skoog basal medium supplemented with 4.5 mg l⁻¹ 2,4-D and 1 mg l⁻¹ BAP was the best medium for callus induction, while the combination of 2 mg l⁻¹ BAP and 1 mg l⁻¹ NAA induced the highest rate of regeneration and development of shoots and roots. This work provides a basis for the breeding of centipedegrass through somaclonal variation and genetic transformation.     

Micropropagation of black pepper (Piper nigrum Linn.) through shoot tip cultures

Summary The morphogenetic potential of shoot tip explants of black pepper (Piper nigrum) was investigated and an effective multiple-shoot propagation method is described. Various combinations of media, growth regulators and sterilization treatments were compared. Problems with establishment in tissue culture sometimes occurred, probably caused by endogenous pathogens associated with tissue exudates. The best establishment and proliferation of shoot tip explants was obtained on MS medium containing 1.5 mg l^–1 BAP alone; subsequent growth and development of lateral branches was best on media containing 1.5 mg l^–1 BAP plus 3.0 mg l^–1 IBA. Adenine sulphate inhibited the number of explants showing regeneration but increased the number of shoot buds per regenerating explant. Shoots were rooted on a 50% strength medium containing 1mg l^–1 NAA.Abbreviations AdSO₄ adenine hemisulphate - BAP 6-benzylaminopurine - IBA indole-3-butyric acid - NAA napthaleneacetic acid     

Petal: a reliable explant for direct bulblet regeneration of endangered wild populations of <Emphasis Type="Italic">Fritillaria imperialis</Emphasis> L.

Wild populations of Fritillaria imperialis L. are facing extinction and need urgent conservation. This paper presents an efficient system for in vitro direct bulblet regeneration of these populations by petal culturing of flower buds. Petals at different developmental stages, green-closed flower bud (before nectar secretion) and red-closed flower bud (beginning of nectar secretion), were used as explants, and the effects of various proportions of cytokinin to auxin on direct bulblet regeneration pathway were evaluated. More explants switched on direct regeneration pathway in combination of auxins (0.6 mg l⁻¹ NAA + 0.4 mg l⁻¹ IAA) with higher level of cytokinin (1 mg l⁻¹ BAP). In contrast, auxins (0.6 mg l⁻¹ NAA + 0.4 mg l⁻¹ IAA) with lower level of cytokinin (0.1 mg l⁻¹ BAP) produced more bulblets per regenerated explant. In green-closed flower bud stage, direct bulblets regenerated from the end of petal where it was connected to the receptacle, while nectar secretion site was the place of bulblet formation in red-closed flower bud stage. In addition, genotype-dependency of direct bulblet regeneration pathway was investigated by using two different wild populations of Fritillaria imperialis. This plant regeneration procedure was applicable to different Fritillaria genotypes and regenerated bulblets were normal.     

In vitro Clonal Propagation and Organogenesis in Spilanthes acmella (L) Murray: A Herbal Pesticidal Plant of North-East India

Multiple shoots were regenerated in MS medium using different concentrations of BAP and Kn and different combinations of BAP with IAA, NAA and IBA. Highest multiplication of shoots was obtained with BAP (0.75 mg l^?1) with 28.4 shoots per explant after 60 days of culture. Shoots rooted best on IBA (0.5 mg l^?1), numbering 48.8 per explant. Organogenesis was maximum in callus cultured on MS medium supplemented with BAP (2.0 mg l^?1) and IAA (1.0 mg l^?1).     

In vitro differentiation and plant regeneration of Albizia chinesis (Osb.) Merr

Summary Petiolar and distal cotyledonary segments (PCS and DCS) of Albizia chinensis were cultured on Murashige and Skoog's (MS; 1962) medium and induced to form adventitious shoot buds in the presence of either cytokinins 6-benzylamino purine (BAP), kinetin (KN) or thidiazuron (TDZ). Superiority of BAP in inducing shoot bud and differentiation was observed. PCS was more morphogenic to shoot bud differentiation than DCS. TDZ was highly effective in inducing shoot buds, but arrested shoot growth, while KN produced more callus during differentiation of shoots. Rapid and high rate of shoot multiplication per explant was achieved through subculture in MS medium containing BAP (1.0 mg l⁻¹) and indole-3-acetic acid (IAA) (0.5 mg l⁻¹). BAP at low concentration was required to enhance shoot multiplication and elongation. Successful rooting of regenerated shoots was carried out in a two-step culture procedure in MS media with indole-3-butyric acid (IBA) (2.0 mg l⁻¹) and subsequent subculture in IBA-free medium.     

In vitro regeneration of Ruscus hypophyllum L. plants

Callus cultures were established from stem explants of Ruscus hypophyllum on a modified basal medium of Murashige and Skoog (1962) supplemented with 1 mg l^-1 2,4-D+0.1 mg l^-1 BAP. The optimal 2,4-D concentration for promoting shoot bud formation and growth was 0.05 mg l^-1 along with 0.5 mg l^-1 BAP. Sixty percent of rootless shoots produced flowers on the regenerating medium. Rooting was induced when shoots were transferred to half strength MS inorganic salts supplemented with 2 mg l^-1 IBA. Eighty percent of plants transferred to soil have survived.     

The role of GA,IAA and BAP in the regulation of <Emphasis Type="Italic">in vitro</Emphasis> shoot growth and microtuberization in potato

The effect of auxin, GA and BAP on potato shoot growth and tuberization was investigated under in vitro condition. The shoot length of potato explants increased with the increasing of concentrations (0.5 – 10 mg dm⁻³) of IAA treatment especially with the addition of GA₃ (0.5 mg dm⁻³), but was inhibited by BAP (5 mg dm⁻³). The root number and root fresh weight of potato explants increased with the increasing of IAA levels either in the presence of GA₃ (treatment IAA+GA) or not (IAA alone). However, no root was observed in the treatment IAA+BAP, instead there were brown swollen calli formed around the basal cut surface of the explants. The addition of GA₃ remarkably increased the fresh weight and diameter of calli. Microtubers were formed in the treatments of IAA+BAP and IAA + GA + BAP but not observed in the treatments of IAA alone or IAA + GA. IAA of higher concentrations (2.5 – 10 mg dm⁻³) was helpful to form sessile tubers. With the increasing of IAA levels, the fresh weight and diameter of microtubers increased progressively. At 10 mg/L IAA, the fresh weight and diameter of microtubers in the treatment of IAA + GA + BAP were 409.6 % and 184.4 % of that in the treatment of IAA + BAP respectively, indicating the interaction effect of GA and IAA in potato microtuberization.     

Organogenesis and embryogenesis inHelianthus tuberosus and in the interspecific hybridHelianthus annuus ×Helianthus tuberosus

A method of plant regeneration from cotyledons ofHelianthus tuberosus, Helianthus annuus ×Helianthus tuberosus and for the backcross of the interspecific hybrids onH. annuus was developed. Induction of somatic embryogenesis and plantlet regeneration from anther culture of the interspecific hybridsH. annuus ×H. tuberosus is reported.Cotyledons were cultured on Murashige and Skoog basal medium (MS) supplemented with indole-3-acetic acid (IAA) and 6-furfurylaminopurine (kinetin) or N⁶-benzylaminopurine (BAP). Shoot regeneration occurred on most of the media tested, but the best results were obtained on media with a high concentration of cytokinins (BAP or kinetin: 4 mg l^–1) and lower concentration of auxin (IAA: 0.5–1 mg l^–1).Embryogenic callus and adventitious buds were initiated from only two anthers of the hybridH. annuus ×H. tuberosus cultured on the MS medium containing BAP (0.2 mg l^–1) and 1-naphtalenacetic acid (NAA: 0.1 mg l^–1). Prolonged culture of these embryogenic calli and buds on the original medium with successive subculture on MS basal medium without growth regulators resulted in embryo formation and shoot differentiation. The plantlets, after rooting, were established in soil.     

In Vitro Regeneration from Seedling Organs of Brassica oleracea var. italica Plenck cv. Green Comet I. Effect of Plant Growth Regulators

The calabrese cultivar Brassica oleracea var. italica cv. GreenComet was used in a study of the effects of exogenous hormoneson the growth and differentiation of seedling organs in vitro.Four types of explants were tested: hypocotyl segments, rootsegments, primary leaf discs and cotyledon discs. These explantswere incubated on media containing factorial combinations ofBAP x IBA, BAP x NAA, KN x IBA and KN x NAA (all at 0, 0.1,10 and 10.0mg l^–1). Hypocotyls were the most regenerativeexplants; shoot production was favoured by cytokinin: auxinratios greater than one and was decreased by IBA at 10 mg l^–1when callus was produced. Shoot formation from root explantsoccurred either in the absence of hormones or with low concentrations;no shoot was produced when any hormone was present at 10 mgl^–1. In contrast, shoot production from primary leaf diseswas favoured by high concentrations of both auxin and cytokininwith the combination of BAP and IBA the most effective. Shootproduction from cotyledon discs was sporadic with no consistentresponse on any auxin/cytokinin combination. After further experimentson the optimization of hormone concentration, the followingcombinations were chosen as allowing reliable regeneration:0.1 mg l^–1 BAP+0.1mg l^–1 IBA for hypocotyl segments,0.075 mg l^–1 KN +0.025 mg l^–1 IBA for root segments,and 5.0 mg l^–1 BAP+5.0 mg l^–1 IBA for leaf discs. Brassica oleracea var. italica, calabrese, tissue culture, seedling, auxin, cytokinin