In vitro shoot multiplication and plantlet regeneration from nodal explants of <Emphasis Type="Italic">Cassia angustifolia </Emphasis>(Vahl.): a medicinal plant

An efficient, rapid and reproducible plant regeneration protocol was successfully developed for Cassia angustifolia using nodal explants excised from 14-day-old aseptic seedlings. Of the two cytokinins, 6-benzyladenine (BA) and thidiazuron (TDZ) evaluated as supplements to Murashige and Skoog (MS) medium, TDZ at an optimal concentration of 5.0 μM was effective in inducing multiple shoots. The highest rate of shoot multiplication was achieved on MS medium supplemented with 5.0 μM TDZ and 1.0 μM indole-3-acetic acid (IAA) at pH 5.8. The regenerated shoots when subcultured on hormone free MS medium considerably increased the rate of shoot multiplication and shoot length by end of fourth subculture passage. Rooting was achieved on the isolated shoots using MS medium with 60 μM indole -3- butyric acid (IBA) and 1% activated charcoal for 1 week and subsequently transferring the shootlets to half strength MS liquid media without IBA and activated charcoal. The in vitro raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in greenhouse.     

Comparative analysis of the differential regeneration response of various genotypes of <Emphasis Type="Italic">Triticum aestivum</Emphasis>, <Emphasis Type="Italic">Triticum durum</Emphasis> and <Emphasis Type="Italic">Triticum dicoccum</Emphasis>

An efficient genotype independent, in vitro regeneration system was developed for nine popular Indian wheat cultivars, three each of Triticum aestivum L. viz., CPAN1676, HD2329 and PBW343, Triticum durum Desf. viz., PDW215, PDW233 and WH896, and Triticum dicoccum Schrank. Schubl. viz., DDK1001, DDK1025 and DDK1029, by manipulating the concentration and time of exposure to the growth regulator, thidiazuron (TDZ). A total of 18 (for immature inflorescence and embryo explant) and six (for mature embryo explant) different combinations of growth regulators were tried for callusing and regeneration, respectively. Media combination with low concentration of TDZ (2.2 μM) in combination to auxin and/or cytokinin (depending upon culture stage), was found to be effective for immature and mature explants. Compact, nodular and highly embryogenic calli were obtained by using immature embryo, immature inflorescence and mature embryo explants, and regeneration frequency up to 25 shoots/explant with an overall 80% regeneration was achieved. Comparable regeneration frequency was achieved for mature embryo explants. No separate hormone combination for rooting was required and plantlets ready to transfer to soil could be obtained in a short period of 8–10 weeks. This protocol can be used for raising transgenic plants for functional genomics analysis of agronomically important traits in the three species of wheat.     

High-frequency shoot multiplication in Artemisia vulgaris L. using thidiazuron

An in vitro propagation system for Artemisia vulgaris L., a traditional medicinal plant, has been developed. The best organogenic response, including adventitious shoot number and elongation, was obtained when hypocotyl segments were cultured onto MS medium supplemented with 4.54 μM TDZ (N-phenyl-N′-(1,2,3-thidiazol-yl) urea). Up to 28 shoots formed per explant for an optimal duration of exposure of 48 days. Regenerated shoots formed roots when subcultured onto a medium containing 8.56 μM IAA (indole-3-acetic acid). Healthy plantlets were transferred to a garden soil:farmyard soil:sand (2:1:1) mixture for acclimatization, which was successful, and subsequent maturity was achieved under greenhouse conditions over a six-month period. The survival rate of the plantlets varied under acclimatization. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of A. vulgaris. This optimized protocol has been successfully employed for genetic transformation studies in A. vulgaris, which are currently underway in our laboratory.     

The mode of action of thidiazuron: auxins,indoleamines, and ion channels in the regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.

The biochemical mechanisms underlying thidiazuron (TDZ)-induced regeneration in plant cells have not been clearly elucidated. Exposure of leaf explants of Echinacea purpurea to a medium containing TDZ results in undifferentiated cell proliferation and differentiated growth as mixed shoot organogenesis and somatic embryogenesis. The current studies were undertaken to determine the potential roles of auxin, indoleamines, and ion signaling in the dedifferentiation and redifferentiation of plant cells. E. purpurea leaf explants were found to contain auxin and the related indoleamine neurotransmitters, melatonin, and serotonin. The levels of these endogenous indoleamines were increased by exposure to TDZ associated with the induction of regeneration. The auxin-transport inhibitor 2,3,5-triiodobenzoic acid and auxin action inhibitor, p-chlorophenoxyisobutyric acid decreased the TDZ-induced regeneration but increased concentrations of endogenous serotonin and melatonin. As well, inhibitors of calcium and sodium transport significantly reduced TDZ-induced morphogenesis while increasing endogenous indoleamine content. These data indicate that TDZ-induced regeneration is the manifestation of a metabolic cascade that includes an initial signaling event, accumulation, and transport of endogenous plant signals such as auxin and melatonin, a system of secondary messengers, and a concurrent stress response.     

Two-stage culture for producing berberine by cell suspension and shoot cultures of <Emphasis Type="Italic">Berberis buxifolia</Emphasis> Lam

In vitro cultures of Berberis buxifolia were established using thidiazuron (4.5, 23 and 45 mM) or picloram (4 and 40 mM) as plant growth regulators for sustaining growth. For producing berberine, a two-stage culture was performed. In the first step, thidiazuron or picloram were used for biomass production followed by the production stage where benzylaminopurine (4.4 mM) was added as a plant growth regulator. Berberine yields (102 mg g⁻¹ DW) and in vitro shoot cultures (200 mg g⁻¹ DW) were significantly lower than those of whole plants in the field (416 mg g⁻¹ DW). The highest productivity (0.18 mg 1⁻¹ day⁻¹) was attained using picloram (either 4 on 40 mM) in the first stage for producing biomass.     

Direct differentiation of somatic embryos on different regions of intact seedlings of Azadirachta in response to thidiazuron

Direct differentiation of somatic embryos occurs in high-frequency and at high density in response to 1.0 microM TDZ, on different regions-hypocotyl, epicotyl, cotyledonary-node, cotyledons and leaves-of intact seedlings of Azadirachta. One-week-old seedlings on this medium exhibited stress symptoms as visible by the loss of root formation and reduction in the elongation of hypocotyl and epicotyl. Globular somatic embryos were more abundant on hypocotyl, epicotyl, stem tip and leaves. The arrest of embryos at this stage was possibly due to their presence in high density. Well-developed somatic embryos were present on the cotyledons and the cotyledonary-node. These embryos on isolation and transfer to hormone-free medium regenerated readily to form plantlets. The possible role of stress in thidiazuron-induced somatic embryo formation is discussed.     

Correlation of epiphyllous bud differentiation with foliar senescence in crassulacean succulent Kalanchoe pinnata as revealed by thidiazuron and ethrel application

Leaves of Kalanchoe pinnata have crenate margins with each notch bearing a dormant bud competent to develop into a healthy plantlet. Leaf detachment is a common signal for inducing two contrastingly different leaf-based processes, i.e. epiphyllous bud development into plantlet and foliar senescence. To investigate differentiation of bud and its correlation, if any, with foliar senescence, thidiazuron (TDZ), having cytokinin activity and ethrel (ETH), an ethylene releasing compound, were employed. The experimental system was comprised of marginal leaf discs, each harbouring an epiphyllous bud. Most of the growth characteristics of plantlet developing from the epiphyllous bud were significantly inhibited by TDZ but promoted by ETH. The two regulators modulated senescence in a manner different for leaf discs and plantlet leaves. Thus, TDZ caused a complete retention whereas ETH a complete loss of chlorophyll in the leaf discs. In contrast, the former resulted in a complete depletion of chlorophyll from the plantlet leaves producing an albino effect, while the latter reduced it by 50% only. In combined dispensation of the two regulators, the effect of TDZ was expressed in majority of responses studied. The results presented in this investigation clearly show that the foliar processes of epiphyllous bud differentiation and senescence are interlinked as TDZ that delayed senescence inhibited epiphyllous bud differentiation and ETH that hastened senescence promoted it. A working hypothesis to interpret responsiveness of the disc-bud composite on lines of a source-sink duo, has been proposed.     

Shoot regeneration from leaves of Prunus serotina Ehrh. (black cherry) and P. avium L. (wild cherry)

Leaves excised from shoot cultures of Prunus avium cvs. F12/1 and Charger and genotype 1908, and from five genotypes of P. serotina and two hybrids of P. avium×P. sargentii developed shoots on Woody Plant medium (WPM) supplemented with either benzyladenine (BA) or thidiazuron (TDZ). Regeneration in both P. avium 1908 and a genotype of P. serotina was improved using TDZ rather than BA in the medium. Regeneration occurred more frequently in P. serotina if leaves were cultured on medium with WPM rather than modified Driver and Kuniyuki walnut medium. The proportions of leaves that regenerated varied between genotypes of the same species. Regenerated shoots of both P. avium and P. serotina developed into shoot cultures following transfer to the media used to produce the shoot cultures used as explant sources. Received: 10 July 1996 / Revision received: 11 November 1996 / Accepted: 6 January 1997     

Efficient shoot organogenesis of eggplant (Solanum melongena L.) induced by thidiazuron

Morphogenesis from several explant types excised from in-vitro-grown plantlets of a Brazilian eggplant (Solanum melongena L.) variety (F-100) was evaluated in response to thidiazuron (TDZ). Leaves and cotyledons were found to be the most responsive explants. Optimal shoot bud induction rates (75–100 buds/explant) were achieved in the presence of 0.2 μm TDZ. Organogenic calli were transferred to growth regulator free MS medium before shoot excision. Rooting was induced on half-strength MS medium supplemented with 0.6 μm IAA. Received: 1 March 1997 / Revision received: 29 October 1997 / Accepted: 15 November 1997     

Combination of thidiazuron and basal media with low salt concentrations increases the frequency of shoot organogenesis in soybeans [Glycine max (L.) Merr.]

A successful, efficient system for multiple soybean shoot induction of soybean [Glycine max (L.) Merr.] is reported. Multiple shoots were induced from cotyledonary nodes and hypocotyl segments cultured on media supplemented with 2 mg/l thidiazuron (TDZ) or 1.15 mg/l benzyladenine (BA). It was found that TDZ induced adventitious shoots more efficiently than BA and that hypocotyl segments promoted more adventitious shoots than cotyledonary nodes. The optimal TDZ concentrations for shoot organogenesis from hypocotyl segments were between 1 and 2 mg/l. Basal media also influenced the efficiency of shoot organogenesis. The frequency of adventitious shoot formation tended to increase when the salt concentration in the basal media supplemented with 2 mg/l TDZ was reduced. Two media (1/2B5 and 1/2L2) stimulated shoot organogenesis efficiently from hypocotyl segments. This method can thus be advantageously applied in the production of transgenic soybean plants. Received: 3 July 1996 / Accepted: 9 May 1997