Efficient plant regeneration in vitro from red leaf beet via organogenesis

An efficient system for in vitro regeneration of red leaf beet, a variety of leaf beet (Beta vulgaris L. var cicla L.) generally used to decorate parterre and to prepare betacyanin, was developed for the first time in the present study. Shoot tip and petiole explants from the sterile seedlings, precultured on Murashige and Skoog (MS) medium with 15 mg/l 6-benzyladenine (BA) and 3% sucrose at 16 °C for 30 days, could form 81.02 and 17.33% translucent nodular (TN) calli, respectively. All TN calli were able to differentiate into adventitious shoots under the same culture conditions. Each explant with TN callus from the shoot tip and petiole could generate 8.65 shoots on average. It was found that both preculture of sterile seedlings and culture of explants at low temperature (16 °C) were vital for TN callus induction and adventitious bud formation of red leaf beet. The best condition for rooting was 0.5-strength MS medium with 10 g/l sucrose. After being transplanted into soil, plantlets grew well and could flower and bear fruits. Histological observation revealed that TN callus was derived from the cells of vascular tissue of the petiole and that adventitious shoots were formed through organogenesis. The factors influencing in vitro micropropagation are also discussed. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 4, pp. 603–608. This text was submitted by the autors in English.     

In Vitro Shoot Regeneration from Callus, Leaf Axils and Petioles of Sugar Beet (Beta vulgaris L.)

Procedures are described for producing axillary shoots fromseedling apices and adventitious shoots from petioles and leaf-derivedcallus of sugar beet cultivars. The rate of adventitious shootregeneration from petioles was influenced by temperature, BAPconcentration of the medium, and the time in culture of theseedling apices from which the petioles were excised. Petiolesectioning confirmed that adventitious shoots originated inthe sub-epidermal parenchyma. Two distinct types of callus wereproduced from leaf explants, but only white friable callus wascapable of shoot development. This callus developed from browntissue and was composed of thin-walled cells with dense cytoplasmand prominent nuclei. Green compact callus with thick-walledlignified cells developed from green tissue, but did not produceshoots. Successful seed sterilization and shoot regenerationfrom petiole explants and callus was cultivar-dependent. Adventitiousshoots were rooted and successfully transplanted to pottingcompost under glasshouse conditions. Key words: Adventitious shoots, axillary shoots, callus, sugar beet (Beta vulgaris L.)     

Direct Organogenesis from Petiole and Thin Cell Layer Explants in Sugar Beet Cultured In Vitro

Detrez, C., Tetu, T., Sangwan, R. S. and Sangwan-Norreel, B.S., 1988. Direct organogenesis from petiole and thin cell layerexplants in sugar beet cultured in vitro.—J. exp. Bot.39: 917–926. Plant regeneration was obtained by direct bud formation frompetiole as well as from thin cell layer explants taken fromsugar beet (Beta vulgaris L.) plants grown in vitro. The budswere mainly induced in the blade-petiole transition zone ofthe explants. High frequency bud regeneration was observed inpetiole and thin layer explants of 10 different breeding linesof sugar beet tested. Organogenesis resulted when petiole explantsexcised from 8-d-old seedlings grown on half-strength Murashigeand Skoog medium (MS) containing 3.0 mg dm^–3 naphthaleneacetic acid (NAA), 3.0 mg dm^–3 6-benzylaminopurine (BAP)and 1.0 mg dm^–3 2, 3, 5, triiodobenzoic acid (TIBA) werecultured on MS with 3.0 mg dm^–3 NAA and 3.0 mg dm^–3BAP. Thin cell layer strips isolated from shoot apices culturedon MS medium supplemented with 0–9 mg dm^–3 BAP or1.0 mg dm^–3 indolebutyric acid (IBA) formed adventitiousbuds on MS medium containing 0–5 mg dm^–3 NAA + 5.0mg dm^–3 BAP. Histological studies confirmed the sub-epidermalorigin of shoots. Key words: Beta vulgaris, direct organogenesis, in vitro culture, petiole, regeneration, thin cell layer     

Influence of some adjuvants on <Emphasis Type="Italic">in vitro</Emphasis> clonal propagation of male and female jojoba plants

Summary The influence of different adjuvants, activated charcoal (AC), casein hydrolyzate (CH), coconut water (CW), polyvinylpyrrolidone (PVP), and triiodobenzoic acid (TIBA), has been assessed on the shoot production potential of the nodal explants derived from in vitro-raised male and female jojoba (Simmondsia chinensis) shoots. Nodal explants of each sex were cultured separately on Murashige and Skoog medium supplemented with different levels of AC, CW, CH, PVP, and TIBA either alone or along with optimum levels of N ⁶-benzyladenine (BA; 10 μM for male, 20 μM for female). Some differences in response of the explants of both the sexes have been observed in terms of (1) percentage of explants developing shoots, (2) average shoot number, and (3) average shoot length. AC alone proved beneficial for elevating morphogenic response in male as well as female explants in comparison to basal medium or media containing AC and the optimum level of BA. When used alone, CH proved inhibitory for shoot differentiation in both sexes, especially in male explants. Addition of PVP to MS enhanced shoot proliferation in female explants only, but along with BA it increased the response of male explants. BA in combination with different levels of TIBA promoted shoot multiplication in female explants. Thus, explants of both male and female shoots exhibited differential morphogenic behavior under the influence of various adjuvants. However, BA alone proved to be the best for differentiation of shoots in both male (10 μM) as well as female (20 μM) explants.     

Identification of highly regenerative plants within sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) breeding lines for molecular breeding

Summary Development of an efficient transformation method for recalcitrant crops such as sugar beet (Beta vulgaris L.) depends on identification of germplasm with relatively high regeneration potential. Individual plants of seven sugar beet breeding lines were screened for their ability to form adventitious shoots on leaf disk callus. Disks were excised from the first pair of true leaves of 3-wk-old seedlings or from partially expanded leaves of 8-mo.-old plants and cultured on medium with 4.4 μM 6-benzylaminopurine for 10 wk. At 5 wk of culture, friable calluses and adventitious shoots began to develop. Rates of callus and shoot formation varied between breeding lines and between individual plants of the same line. Line FC607 exhibited the highest percentage (61%) of plants that regenerated shoots on explants. Among the plants with a positive shoot regeneration response, line FC607 also had the highest mean number (8.3±1.1) of shoots per explant. Individual plants within each line exhibited a wide range of percentages of explants that regenerated shoots. A similar variation was observed in the number of shoots that regenerated per explant of an individual plant. No loss of regeneration potential was observed on selected plants maintained in the greenhouse for 3 yr. Regenerated plants exhibited normal phenotypes and regeneration abilities comparable to the respective source plants. Based on our results, it is imperative to screen a large number of individual plants within sugar beet breeding lines in order to identify the high regenerators for use in molecular breeding and improvement programs.     

Comparative adventitious shoot induction in kentucky coffeetree root and petiole explants treated with thidiazuron and benzylaminopurine

Summary Adventitious shoot induction and elongation was compared between root and petiole explants of Kentucky coffeetree (Gymnocladus dioicus L.) explants treated with a factorial combination of benzylaminopurine (BA) and thidiazuron (TDZ). Petiole explants initiated more adventitious shoots compared to root explants. Up to 83% of petiole explants initiated shoots compared to 67% of root explants. Maximal shoot induction was approximately 12 or five shoots per responding explant for petiole and root explants, respectively. For both explant types, TDZ was more effective than BA for shoot induction. There was an interaction between BA and TDZ on shoot induction in petiole explants, with the greatest percentage of explants forming shoots and the highest number of shoots initiated on the combination of 0.5 μM TDZ plus 10μM BA and 1.0μM TDZ plus 5 or 10 μM BA. In contrast, increasing concentrations of BA inhibited shoot initiation in root explants with and without TDZ. While BA inhibited shoot initiation in root explants, it promoted shoot initiation in petiole explants. In contrast, TDZ was equally effective at inducing shoots in root and petiole explants. This suggests that root and petiole explants of Kentucky coffeetree could be a useful model system for studying the differences, in apparent mode of action between TDZ and BA on adventitious shoot initiation.     

Efficient plant regeneration from petiole explants of West Indian gherkin (Cucumis anguria L.) via indirect organogenesis

An efficient protocol for in vitro organogenesis was achieved from callus-derived immature and mature petiole explants of West Indian gherkin (Cucumis anguria L.). Calluses were induced from immature petiole explants excised on 7-day-old in vitro seedlings and mature petiole explants of 40-day-old in vivo plants. The maximum frequency of immature petiole explants (98.0 %) and mature petiole (91.5 %) produced green, compact organogenic callus in Murashige and Skoog (MS) medium with Gamborg (B5) vitamins containing 30 g l^?1 sucrose, 8.0 g l^?1 agar and 4.0 μM naphthalene acetic acid (NAA) with 2.0 μM benzyl amino purine (BAP) after two successive subculture at 11 days interval. Adventitious shoots were produced from the organogenic callus when it was transferred to MSB₅ medium supplemented with 3.0 μM TDZ, 1.0 μM NAA and 0.05 mM L-glutamine with shoot induction frequency of immature petiole 45 shoots and mature petiole 40 shoots per explant. The shoots were excised from callus and elongated in MSB₅ medium fortified with 3.0 μM gibberellic acid (GA₃). Then elongated shoots were rooted in half strength MSB₅ medium supplemented with 3.0 μM indole 3-butyric acid (IBA). Histological analyses of the regeneration process confirmed the indirect organogenesis pattern. Plantlets with well-developed shoot and root systems were successfully acclimatized (95 %) in winter season and exhibited normal morphology and growth characteristics. The survival percentage differed with seasonal variations.     

Micropropagation of <Emphasis Type="Italic">Scabiosa caucasica</Emphasis> Bieb. Cv. Caucasica blue

Summary Micropropagation of Scabiosa caucasica cv. Caucasica Blue was achieved by culturing, separating axillary and adventitious shoots, or node sectioning on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA). The highest frequency of adventitious shoots regenerated from nodal or internodal explants and leaf blade (with or without petiole) appeared to occur on MS medium with 4.4 and 18 μM BA, respectively. Addition of 0.19 or 1.9 μM α-naphthaleneacetic acid to the BA-containing medium promoted callus formation and reduced shoot organogenesis. During micropropagation, shoot nodal explants derived from in vitro shoots cultured on MS medium supplemented with 4.4 μM BA yielded 8.9 shoots per explant within 40 d after culture initiation.     

A two-stage pretreatment of seedlings improves adventitious shoot regeneration in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.)

The effects of a two-stage pretreatment of seedlings on the subsequent shoot regeneration capacity were investigated. Pretreated seedlings were obtained by germinating seeds on three different germination media and then further culturing on six different growth media. Lamina and petiole explants of two sugar beet (Beta vulgaris L.) breeding lines were then excised from the pretreated seedlings and cultured on five different shoot regeneration media. In both breeding lines, petiole explants produced significantly more shoots than lamina explants with higher frequencies of organogenic capacities; petiole explants of the lines M1195 and ELK345 produced a mean of 2.1 and 2.7 shoots per explant while their lamina explants produced 1.5 and 2.2 shoots per explant, respectively. A genotypic variation was evident as the line ELK345 was more productive for shoot development from both types of explants. In overall comparisons of different germination, growth and regeneration media, germination medium was most effective when supplemented with 0.5 mg/l 6-benzyladenine (BA) while both growth and regeneration media were most productive when contained a combination of 0.25 mg/l BA and 0.10 mg/l indole-3-butyric acid (IBA). Of all the treatments tested, the highest mean number of shoots per explant (8.3 shoots) and frequency of organogenic explants (75.6%) were obtained on regeneration medium supplemented with 0.25 mg/l BA and 0.10 mg/l IBA when petiole explants of the line ELK345 were excised from the seedlings that had been germinated on medium containing 0.5 mg/l BA followed by further growth on medium containing 0.25 mg/l BA and 0.10 mg/l IBA.     

The Effect of 2,3.5 Triiodobenzoic Acid on Caulogenesis in Callus Cultures of Tomato and Pelargonium

Stem explants from winter grown tomatoes cultured on a cytokinin, auxin-free medium, developed one or two adventitious shoots at the top end of the explant. Addition of the auxin transport inhibitor. 2,3,5-triiodobenzoic acid (TIBA) to the medium stimulated caulogenesis with loss of polarity. Callus, initiation in pelargonium and ‘geranium’ petiole explants requires both auxin and cytokinin. On transfer, after callus induction to an auxin-free medium, rhizogenesis occurs in pelargonium cultures followed by caulogenesis. Few shoots develop and unless these are removed, further caulogenesis is suppressed. Bud-like structures were formed in the callus. Subculture on auxin-free medium containing cytokinin and TIBA resulted in shoot formation from these bud-like organs. An analogy with apical dominance is suggested. In ‘geranium’ callus, shoots developed with a low frequency (c. in 2% of the cultures): caulogenesis was increased to 80% when calli were subcultured from auxin-free, cytokinin medium after green nodule formation to cytokinin-TIBA medium. Histological studies of green nodules in ‘geranium’ callus indicated a variation in morphological development within and between nodules. It is suggested that auxin synthesis may occur at some microscopic stage in morphogenesis in ‘geranium’ cultures which suppresses further caulogenesis. This may be overcome by the addition of TIBA to the medium at the appropriate stage in morphogenesis. The possible interaction of endogenous auxin in morphogenesis is discussed.     

Tissue culture response of Beta germplasm: callus induction and plant regeneration

Callus cultures of 18 sugarbeet (Beta vulgaris) lines, two accessions of B. maritima and a B. macrocarpa accession were initiated from aseptically germinated seeds. Plant regeneration through organogenesis was obtained either on MS or B5 medium containing various concentrations and combinations of naphthaleneacetic acid (NAA), 6-benzylaminopurine (BAP), 2,3,5-triiodobenzoic acid (TIBA) and abscisic acid (ABA). Genotypes differed in their abilities of callus formation and regeneration: seven out of 18 sugarbeet lines, and an accession of B. maritima were capable of regenerating plantlets. Our data also indicated that 2 M TIBA promoted morphogenesis from callus culture in the presence of 5 M BAP.     

Regeneration of plantlets from leaf and petiole explants of Pelargonium x hortorum

Summary The in vitro plant regeneration potential of vegetatively propagated geraniums (Pelargonium x hortorum) has been investigated. Using various combinations of growth regulators and a choice of different explants, a regeneration protocol has been developed to raise in vitro plantlets from young petiole and leaf explants from three different cultivars of geraniums. In all three cultivars, very young petiole explants exhibited a higher regeneration potential as compared with leaf explants. Regeneration efficiencies were found to be highly dependent on the cultivar, with cv. Samba showing the highest regeneration potential, followed by cvs. Yours Truly and then Sincerity. Samba also showed the highest number of shoots from both the petiole [57 shoot buds per petiole explant in the presence of 3 μM zeatin and 1 μM indole-3-acetic acid (IAA) and leaf explants (43 shoots per leaf explant with 10 μM zeatin and 2 μM IAA). Shoot buds transferred to Murashige and Skoog (MS) medium supplemented with 0.44 μM N⁶-benzyladenine and 0.11 μM IAA grew vigorously and attained 1–2 cm in length in 3–4 wk. These shoots rooted with 100% efficiency on MS basal medium, and plants developed that showed normal growth and flowering under greenhouse conditions.     

In vitro propagation of the alkaloid producing plant Datura insignis Barb. Rodr.

A method is presented for the in vitro propagation of Datura insignis Barb. Rodr. Nodal explants were cultured on Murashige and Skoog medium supplemented either with BA alone or in combination with 2,4-D or IAA. Shoot multiplication and elongation were obtained in various growth regulator concentrations. Best results were obtained in a medium with 1.0 mgl^-1 of BA. Individual shoots were excised and transfered to growth regulator-free medium for rooting. Additional multiplication was obtained by single-node culture using explants from these in vitro rooted shoots. Rooted plantlets were successfully grown in soil.     

In vitro culture of petioles and intact leaves of sugar beet (Beta vulgaris)

Methods are described for obtaining explants which produce adventitious shoots, for subsequent stimulation of rooting and then transplanting using six commercial sugar-beet cultivars. The rate of adventitious shoot regeneration from petioles or intact leaf explants was affected by the source of donor plants, cytokinin type (BAP or Kin) and concentration and cultivar. Increasing the sucrose concentration of the medium from 3% to 5% or 8% had no apparent effect. Adventitious shoots could be produced directly from callus formed on the base of the petioles. In general adventitious shoots were produced on either the concave surface of the petiole or from the callus, occasionally simultaneously on both, and on the convex surface of the petiole in intact leaf explants. The highest rooting rate with 3% sucrose and 1.0 mg l^–1 NAA was obtained using half-strength MS medium. There was considerable variation in the propagules from petioles or callus indicating that this system may provide valuable somaclonal variation.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

Effect of genotype,explant type and growth regulators on organogenesis in Morus alba

Plantlets of the mulberry (Morus alba L. vars. Chinese White, and Kokuso-27) were produced from callus cultures. For callus induction, leaf, internodal segments, and petiole explants of Chinese White, Kokuso-27 and Ichinose varieties were grown on MS basal medium fortified with 2,4-D and 6-benzylaminopurine (BA). Callogenesis was dependent on the nature of explant used, the genotype and growth regulators supplemented in the medium. Leaves were the best explant type used for callus induction. Best callogenesis was obtained on MS medium containing a combination of 1 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ BA (95-100%). Calluses formed shoots on MS medium supplemented with 1 mg l⁻¹ BA. Supplementation with 0.1 mg l⁻¹ 2,3,5-triiodobenzoic acid (TIBA) in this medium enhanced shooting response. Presence of TIBA in the medium also improved the long-term organogenic potential of the callus. Regenerated shoots produced roots on Murashige & Skoog (MS) medium containing either 0.5 mg l⁻¹ indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). Seventy percent of the rooted plants were established in the field where they are performing well. This revised version was published online in June 2006 with corrections to the Cover Date.     

Callus and Root Formation in Explants of Beta vulgaris

Callus from hypocotyl and petiole explants and anthers of Beta vulgaris L. was initiated on a defined agar medium containing IAA and kinetin. The cultures were kept in light (18 hours a day) at 27C for 5 weeks. Differences in dry matter production and root initiation were found between hypocotyl and petiole explants. No shoots were formed. Callus formation in anthers was very rare.     

TDZ-induced high frequency plant regeneration through multiple shoot formation in witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.)

A very efficient and rapid regeneration system via multiple shoot formation was developed for Cichorium intybus L. when leaf explants excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. In a comparison of leaf lamina and petiole explants, lamina explants produced over three times more shoots than petiole explants, with a mean of 7.5 shoots compared to 2.4. Of the combinations of KIN/IAA, KIN/NAA, BAP/IAA, or BAP/NAA, 0.5 mg l⁻¹ KIN combined with 0.3 mg l⁻¹ IAA was the most effective, producing a mean of 19.7 shoots per lamina explant while the control treatment involving no plant growth regulators produced no shoots at all. When either cytokinin was used alone, BAP was found nearly twice more successful than KIN. However, the most effective treatment of all was the combination of 0.01 mg l⁻¹ TDZ and 1.0 mg l⁻¹ IAA, producing as many as 35.8 shoots per lamina explant. This rate of shoot regeneration is remarkably higher than those previously reported for C. intybus, most likely due to the highly inductive effect of TDZ, which was tested for the first time in this species. Rooting of the shoots was readily achieved on medium containing different concentrations of IAA or IBA. IAA was more effective than IBA and resulted in the highest frequency of shoots that rooted (100%) and mean number of roots per shoot (4.2) when used at 0.5 mg l⁻¹. Hardening off process resulted in a production of more than 80% healthy plantlets.     

In vitro organogenesis from leaf explants of Annona squamosa Linn

Multiple shoot formation was induced from excised leaf explants of Annona squamosa Linn. (custard apple) seedlings on a Murashige and Skoog basal medium containing benzylaminopurine and kinetin. Various auxins in combination with the above medium produced callusing of the explants. In an investigation of environmental factors affecting shoot induction it was seen that the maximum number of shoots were obtained using the leaf base with petiole at a temperature of 27°C and a light intensity of 1000 lux. Roots were initiated erratically when individual shoots were treated with an auxin and then transferred to an auxin free medium. The process of the development of adventitious buds in leaf culture was analysed histologically.NCL Communication No. 3104.     

Indirect shoot organogenesis from leaves of Dieffenbachia cv. Camouflage

A novel protocol for indirect shoot organogenesis of Dieffenbachia cv. Camouflage was established using leaf explants excised from in vitro shoot cultures. The frequency of callus formation reached 96% for explants cultured on Murashige and Skoog (1962) basal medium supplemented with 5 μM thidiazuron and 1 μM 2,4-dichlorophenozyacetic acid. The number of shoots regenerated was high, with up to 7.9 shoots produced per callus cultured on basal medium supplemented with 40 μM N ⁶-(Δ²-isopentenyl)adenine and 2 μM indole-3-acetic acid. Regenerated shoots rooted well in a soilless substrate, acclimatized ex vitro at 100%, and grew vigorously under shaded greenhouse conditions. Somaclonal variations in leaf variegation, color, and morphology have been observed in regenerated plants.     

adventitious shoot regeneration from petiole explants of Heracleum candicans wall

Summary A method for adventitious shoot induction from petiole explants of Heracleum candicans is reported. Shoot buds were induced on Murashige and Skoog (MS) medium with 4.4μM 6-benzylaminopurine (BA) and 1.1 μM 2,4-dichlorophen-oxyacetic acid (2,4-D). A wound response in the presence of BA and 2,4-D at the time of culture was necessary for inducing shoot buds. The shoot bud regeneration was significantly influenced by size, type and orientation of explants on the culture medium. These shoot buds developed into 4–5 cm shoots upon transfer to a medium containing 1.1μM BA and 0.5 μM α-naphthaleneacetic acid (NAA). The regenerated shoots formed rooted plantlets on MS medium supplemented with 4.9 μM indole-3-butyric acid (IBA). About 15 plants were established in the field for further evaluation.