Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

Regeneration of haploid plants from isolated microspores of asparagus (Asparagus officinalis L.)

High percentages of micro-calli and micro-derived embryos were produced from isolated asparagus microspores at late uninucleate stage on MS liquid medium supplemented with 1.0 mg l^–1 2,4-D and 0.5 mg l^–1 BA. Two types of calli, namely compact callus (CC) and loose callus (LC), were found. Plantlets were regenerated via organogenesis, when these calli were transferred onto MS solid medium supplemented with 1.0 mg l^–1 BA and 0.2 mg l^–1 IBA 6 weeks. Embryos were produced from liquid cultured microspores, or from solid cultured micro-calli. The frequencies of haploid plant production from organogenesis and embryogenesis were compared. Effects of plant growth regulators on callus production, plantlet regeneration, and haploid plant production were tested. The combination of BA 1.0 mg l^–1 and IBA 0.2 mg l^–1 resulted the highest precentage of haploid plant production (7.7% from CC, 4.3% from LC).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IBA 3-indolybutyric acid - BA 6-binzyladinine - NAA naphtalene acetic acid - MS Murashige and Skoog     

Micropropagation of zedoary (<Emphasis Type="Italic">Curcuma zedoaria</Emphasis> Roscoe) – a valuable medicinal plant

Tissue culture propagation system was developed for zedoary (Curcuma zedoaria Roscoe), a valuable medicinal plant, using rhizome sprout cultures. Shoots were induced from rhizomes on basal MS medium containing 20 g l^–1 sucrose and 5 g l^–1 agar, supplemented with 20 (v/v) coconut water (CW) and benzylaminopurine (BA) concentrations from 0.5 to 5.0 m g l^–1. The excised shoots were subcultured on Murashige-Skoog (MS) medium with 20 (v/v) CW and different concentrations of BA and kinetin (Kin), either alone or in combination with indolebutyric acid (IBA) or naphthaleneacetic acid (NAA). MS medium with 20 (v/v) CW, 3 mg l^–1 BA, and 0.5 mg l^–1 IBA resulted in a multiplication rate per shoot; 5.6 shoots per explant were obtained on average after 30 days of culture. Well-developed shoots (30–40 mm in length) were rooted on MS medium containing 20 g l^–1 sucrose and 8 g l^–1 agar, supplemented with 20 (v/v) CW and 2 mg l^–1 NAA. More than 95 of the rooted plants were established in pots after hardening.     

Plant regeneration from cell suspension-derived protoplasts of Saintpaulia ionantha Wendl

Summary Friable calli were induced on leaf segments of Saintpaulia ionantha Wendl. on B5 medium containing 1 mg l^–1 2,4-D and 2 g l^–1 casein hydrolysate. Cell suspension cultures were readily established from these friable calli and protoplasts could be isolated from the cells with yields of 1–3×10⁷/g f. wt.. By culturing in 0.1 % gellan gum-solidified B5 medium supplemented with 1 mg l^–1 2,4-D and 0.1 M each of sucrose and mannitol at a density of 1×10⁵/ml, the protoplasts divided within 6 days and formed macro-colonies after 2 months of culture. Shoot regeneration from protoplast-derived calli was obtained by sequential treatment of the calli with plant growth regulators: initially with 1 mg l^–1 each of NAA and BA for 2 months followed by 0.01 mg l^–1 NAA and 5 mg l^–1 BA for 4 months. Regenerated plants were established after rooting of the shoots on half-strength MS medium, and successfully transferred to the greenhouse. The regenerated plants grew into flowering stage and showed the same phenotype as the parent plant.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - f. wt fresh weight - MES 2-(N-morpholino)-ethanesulfonic acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid - PE plating efficiency     

Callus induction and high-frequency plant regeneration from hypocotyl and cotyledon explants of Arctium lappa L.

Summary This study describes a protocol for plant regeneration from cultured seedling explants of Arctium lappa. Hypocotyls and cotyledons of A. lappa were induced to form callus by culturing on Murashige and Skoog (MS) medium supplemented with 2.0mg l^&#x2212;1 2,4-dichlorophenoxyacetic acid and 0.5&#x2013;2.0 mg l^&#x2212;1 benzyladenine (BA). Formation of adventitious buds could be induced from calluses or explants directly by culturing on MS medium containing 1.0&#x2013;2.0 mg l^&#x2212;1 &#x03B1;-naphthaleneacetic acid (NAA) and 0.5&#x2013;2.0 mg l^&#x2212;1 BA. These regenerated shoots were rooted on MS medium with 1.0 mg l^&#x2212;1 indole-3-butyric acid or indole-3-acetic acid in combination with 1.0 mgl^&#x2212;1 NAA. The regenerated plants acclimatized in soil were normal morphologically and in growth characters. They flowered and set seeds in the following year after acclimatization.     

Effects of foliar applied gibberellic acid and benzyladenine upon yield components in sunflower (Helianthus annuus L.)

Sunflower yield is determined by seed number/m^–2 and by achene weight. Frequently, a high percentage of empty achenes in the inner portion of the capitulum, probably due to a reduced vascularization of that section of the flower head, decreases final yield. The objective of the present research is to determine if foliarly applied gibberellic acid (GA) and benzyladenine (BA) can enhance the vascularization in the inner portion of the capitulum, improving photoassimilate translocation. Field experiments were conducted during 1989/90 with hybrid SPS 894 and during 1990/91 with hybrid ACA 882. GA (150 mg/l^–1), BA (150 and 250 mg/l^–1) and GA 150+BA 150 mg/l^–1 each were foliarly applied 20, 40, or 60 days after emergence. For both seasons and hybrids plant growth regulator (PGRs) applications significantly reduced the percentage of empty achenes, increased achene weight, achene weight (× 1000) and achene number in the inner portion of the capitulum and in the middle and outer portion during 1990/91. A 25% increase in seed yield was achieved due to PGR application and the capitulum partition index (achene weight/receptacle weight^–1, CPI) was significantly increased due to an improvement in photoassimilate distribution. A distribution model was derived showing that preferential allocation of photoassimilates in the outer portion of the capitulum can be modified by PGR application, demonstrating that photoassimilate distribution is under hormonal control.Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET)     

Induced Activity of Superoxide Dismutase and Peroxidase of in vitro Plants by Low Concentrations of Ethanol

A rapid micropropagation protocol was established for Aloe vera L. var. chinensis (Haw.) Berger, Chinese Aloe. The effects of three factors, namely BA, NAA and sucrose, on bud initiation were evaluated by L₉ (3⁴) orthogonal design. The variance analysis of the experimental results showed that the actions of the three factors were all considerable. Among the three factors, sucrose was the most important for bud initiation followed by BA, and NAA had the weakest effect. The best medium for bud initiation was semi-solid MS supplemented with 2.0 mg l^–1 BA, 0.3 mg l^–1 NAA, 30 g l^–1 sucrose and 0.6 g l^–1 PVP (pH 5.8), on which Chinese aloe could multiply 15 times in 4 weeks. Some shoots rooted spontaneously on 1/2 strength MS medium, but the rooting percentage was improved in the presence of 0.2 mg l^–1 NAA. Rooted plantlets were acclimatized to greenhouse conditions. The young plantlets from tissue culture were transplanted successfully. In vitro propagation can be a useful tool in the conservation of this endangered medicinal species.     

Induction of haploid callus and embryogenesis in in vitro cultured anthers of mulberry (Morus indica)

Anthers of Morus indica L., with microspores at the uninucleate stage were cultured; and the influence of temperature and kinetin pretreatment on induction of androgenic calluses was examined. The effects of various pretreatments revealed that 24 h cold pretreatment increased the percentage of cultures inducing callus. First microspore division was observed after 16 to 20 days of culture. Th anthers split and developed embryogenic calluses on MB medium supplemented with NAA (0.5 mg l^–1 and BA (1.0 mg l^–1)) using 8% sucrose. Rhizogenesis was induced on medium supplemented with NAA and BA (each 0.5 mg l^–1) with reduced myo-inositol (75 mg l^–1). Cytological study of induced roots confirmed the haploid nature of calluses. Different type of embryos were initiated upon transfer of calluses to medium supplemented with NAA, BA (each 0.5 mg l^–1), 2,4-d (1.0 mg l^–1) and PVP (600 mg l^–1). These embryoids further developed roots on removal of 2,4-d from the medium and developed precociously without developing cotyledons and formed elongated shoots.Abbreviations BA 6 benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - FAA formalin: Acetic acid: Alcohol - GA₃ gibberellic acid - IBA indole-3-butyric acid - MB modifed Bourgin (Qian et al., 1982) - NAA 1-naphthalene acetic acid - PVP polyvinylpyrrolidone - RFS-135 rainfed selection 135 - SE standard error     

Plant regeneration from suspension cultures of Hosta sieboldiana

Systems for establishing suspension cultures and for inducing plant regeneration from these cultures for the Liliaceous ornamental plant, Hosta sieboldiana (Lodd.) Engl. have been developed. Pale-yellow and nodular calluses were induced from more than 20% of scape segments on MS medium containing 1 mg l^–1 picloram (PIC), 30 g l^–1 sucrose, and 2 g l^–1 gellan gum. Upon transfer of calluses to the same medium lacking gellan gum, stably-growing suspension cultures were established after 1 month. Suspension cell clusters regenerated a large number of adventitious shoots following transfer to MS media containing 0.1 mg l^–1 NAA in combination with either BA or TDZ. Over 20 shoots per 0.3 g FW of cell clusters were obtained on media containing 0.1 mg l^–1 NAA and either 1 or 5 mg l^–1 TDZ. Shoots rooted easily on plant growth regulator (PGR)-free MS medium, and plantlets were successfully transferred to soil. Plants showed no visible morphological alterations and maintained the diploid level as indicated by flow cytometric analysis.     

Plant regeneration through somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis>

A new method was established for somatic embryogenesis and plant regeneration from callus cultures of Dioscorea zingiberensis C.H. Wright. Primary callus was induced by culturing stems, leaves and petioles on Murashige and Skoog (MS) medium supplemented with 0.5–2.0 mg l^–1 N⁶-benzyladenine (BA) and 0–2.0 mg l^–1 -naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) for 1 month. The highest frequency (87%) of callus formation was achieved from stem explants treated with 0.5 mg l^–1 BA and 2.0 mg l^–1 2,4-D. Somatic embryos were obtained by subculturing embryogenic calli derived from stem explants on MS medium supplemented with 2.0–4.0 mg l^–1 BA and 0–0.4 mg l^–1 NAA or 2,4-D for 3 weeks. The optimum combination of 4.0 mg l^–1 BA and 0.2 mg l^–1 NAA promoted embryo formation on one-third of the calli. After a further month of subculture on the same medium, mature embryos were transferred to MS medium supplemented with 0–4.0 mg l^–1 BA, NAA or indole-3-butyric acid (IBA) for further development of plantlets and tuber formation. Plant growth regulators had a negative effect on the development of mature embryos.     

In vitro propagation of green-foliaged Dracaena fragrans Ker.

Callus tissue was induced in young stem segments cultured on MS based media supplemented with 0.25–0.5 mg l^-1 2, 4-D. Shoots were differentiated on media containing 0.5–1.0 mg l^-1 BA and 0.5–2.0 mg l^-1 IBA or 0.1–0.2 mg l^-1 NAA. The same media were suitable for shoot multiplication. Shoot elongation and rooting were strongly inhibited by BA and stimulated by auxins IBA and NAA. Medium containing 0.5 mg l^-1 IBA was optimal for rooting. Root elongation was stimulated by light and inhibited in darkness. Transfer of rooted plantlets to outdoor conditions was feasible and special hardening procedures were not required. Among more than 5000 plants produced by this procedure only 9 off-type plants with variegated leaves were found.     

Effects of foliar applied benzyladenine on grain yield and grain protein in wheat (Triticum aestivum L.)

The effects of foliar applications of nitrogen and benzyladenine (BA) on grain yield and grain protein of wheat grown under field conditions were studied over 2 years with 5 cultivars at 2 locations. Nitrogen (N) at 20 kg.ha^–1, and BA at 100 or 800 mg.l^–1 were applied alone or combined at pre and post-anthesis; applications of BA at 8 mg.l^–1 were also made on individual ears in order to study the effect on cell number. Weekly determinations of the chlorophyll content of the flag leaf were conducted after anthesis to study leaf senescence. At harvest, yield, yield components and grain protein percentage were determined. N and BA applications delayed chlorophyll loss in the flag leaf, but modified neither yield nor yield components. Foliarly applied BA increased grain protein in four of the five cultivars tested. It is concluded that delay of the senescence induced by BA might allow more energy to be available for N uptake by the crop leading to an increase in grain protein.Research supported by a CAFPTA grant 1656/86 and by CONICET, PID 30017700/85.CONICETComisión de Investigaciones Cientificas de la Provincia de Buenos AiresInstituto de Fisiologia Vegetal     

Stable genetic transformation of castor (Ricinus communis L.) via Agrobacterium tumefaciens-mediated gene transfer using embryo axes from mature seeds

A protocol for the transformation of castor embryo axes using the pCAMBIA vector 1304 in disarmed Agrobacterium tumefaciens strain EHA105 is presented. Co-cultivated explants were initially subjected to expansion and proliferation on MS medium with 0.5 mg l^–1 TDZ followed by three cycles of selection on medium with 0.5 mg l^–1 BA and increasing concentrations of hygromycin (20–40–60 mg l^–1). Selected shoot clusters were transferred to medium with 0.5 mg l^–1 BA for proliferation and 0.2 mg l^–1 BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium with 2.0 mg l^–1 NAA. The presence and stable integration of the hpt gene was confirmed through PCR, RT-PCR, PCR-Southern blot, sequence analysis, Southern blot analysis and PCR analysis of progeny. Southern blot analysis of the primary transformants showed single copy integration and progeny analysis revealed monogenic inheritance of the introduced gene. This paper reports the first successful attempt at producing transgenic castor.     

Influence of Liquid Pulse Treatment with Growth Regulators on in vitro Propagation of Banana (Musa spp. AAA)

The effect of liquid pulse treatment of growth regulators on in vitro propagation of banana (Musa spp. AAA) was studied. Optimal shoot proliferation rates were achieved due to the pulse treatment of 6-benzylaminopurine (BA) and kinetin combination (1:1) at the concentration of 50 mg l^–1 for 60 min. Similarly high frequency of root induction was obtained due to pulse treatment with a NAA and IBA combination (1:1) at the concentration of 100 mg l^–1 for 60 min.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration from the immature seeds of <Emphasis Type="Italic">Cymbidium faberi</Emphasis>

An in vitro plant regeneration protocol of Cymbidium faberi from immature seeds was established. The immature seeds of 50 days old started to form rhizomes 4 months after they were cultured on hormone free medium. The rhizomes multiplied 5 times when subcultured on the medium containing 1.0 mg l^–1 -naphthalene acetic acid (NAA) for 40 days and more than 90% of the rhizomes initiated shoots within 60 days on the media containing 0.5 or 1.0 mg l^–1 NAA plus 2.0 or 5.0 mg l^–1N⁶-benzylaminopurine (BA). Plantlets were regenerated when the shoots were planted on the basal medium amended with 1 g l^–1 activated charcoal for 50 days and the plantlets grew normally after transplanting.     

In vitro plant regeneration from leaf callus inPiper colubrinum Link

Callus-mediated shoot regeneration from leaf explants ofPhytophthora resistant pepper (Piper colubrinum Link.) is described. The effect of basal media composition and growth regulators onin vitro response of explants was evaluated. Shoot buds were induced and elongated on half-strength MS medium containing 2.0 mg l^–1 BA and 0.5 mg l^–1 NAA , as well as 1.0 mg l^–1 BA and 0.5 mg l^–1 2,4-D. The shoots were rooted in half-strength MS medium with or without IAA or IBA, and then were transferred to soil with 100% survival.     

Effects of Growth Regulators on Direct Flowering of Isolated Ginseng Buds <Emphasis Type="Italic">in vitro</Emphasis>

An in vitro method for obtaining gingseng inflorescences directly from explants of gingseng (Panax ginseng) is reported. Isolated shoot-buds of somatic embryo-derived plantlets ginseng were used as explants and incubated in B5 medium supplemented with 1 mg l⁻¹ benzyladenine (BA) and 1 mg l⁻¹ gibberellic acid (GA₃). About 15% of the buds flowered directly without developing vegetative organs. Cytokinin was found to be the key factor for inducing these isolated buds to proliferate and flower, but both these processes also occurred when benzyladenine (BA) was replaced by thidiazuron (TDZ). The optimal concentration of TDZ for obtaining the best ratios of bud proliferation and total flowering was 0.1 mg l⁻¹, while the highest number of vegetative shoots was obtained in medium supplemented with 1 mg l⁻¹ GA₃ and 0.1 mg l⁻¹ TDZ. The explant elongated abnormally in the presence of 10 mg l⁻¹ GA₃. Although a low concentration (1 mg l⁻¹) of NAA increased the bud proliferation ratio in the medium supplemented with 0.1 mg l⁻¹ TDZ and 1 mg l⁻¹ GA₃, a high concentration (5 mg l⁻¹) of NAA reduced the bud proliferation ratio and inhibited the flowering.     

Effect of brassinolide on callus growth and regeneration in Spartina patens (Poaceae)

The effect of brassinolide (BL) on cultured calluses of Spartina patens (Ait.) Muhl. (Poaceae), a halophyte monocot was studied. BL at 0.03–0.04 mg l^–1 at fixed concentrations of IAA (0.2 mg l^–1) and BA (3.0 mg l^–1) added in MS medium increased the ratio for fresh weight (CIRFW) to dry weight (CIRDW) by 96–111% and 235–326%. Similarly, in callus regeneration capacity, BL at 0.03 mg l^–1 was most effective, increasing the shoot regeneration ratio (SRR) by 425%. BL at 0.04 mg l^–1 had not such an increasing effect as BL at 0.03 mg l^–1, which increased SRR by 79%. However, BL at 0.005 mg l^–1 promoted regenerated shoot growth most significantly, increasing the shoot height increasing ratio (SHIR) by 395% after a 40-day culture. BL at 0.05 mg l^–1 was least effective in the callus regeneration and regenerated shoot growth, decreasing SRR by 27% and SHIR by 52%. Present results suggest that BL at 0.03 mg l^–1 is suitable for the callus growth and shoot regeneration, while BL at 0.005 mg l^–1 effectively enhanced the regenerated shoot growth.     

Development of morphogenic suspension cultures of garlic (Allium sativum L.)

Rapidly growing, regenerable suspension cultures were obtained from meristem-derived callus cultures of garlic (Allium sativum L.). The liquid culture medium consisted of MS salts, B5 vitamins, 3% sucrose, 1 mg l^–1 naphthalene-acetic acid (NAA) and 2 mg l^–1 6-benzyladenine (BA). The tissue in the suspension culture was yellow, smooth, organized, and proliferated as nodular clumps. Histological examination revealed that these morphogenic clumps had a well-defined epidermis. Following transfer of the morphogenic clumps to an agar-solidified medium, numerous meristems with green leaf primordia were produced.     

Regeneration of bulblets from twin scales ofCrinum macowanii in vitro

Tissue culture techniques were applied to study the regeneration and growth of bulblets from bulb scale segments ofCrinum macowanii Bak. (bush- or march lily)in vitro. Shoots were induced on twin scales taken from the basal plate region of flowering-size bulbs on Murashige and Skoog (MS)-medium containing 0–20 mg l^–1 NAA and BA and a modified MS medium (MMS medium) containing 1.25 mg l^–1 ancymidol (A-Rest^TM), 0.1 mg l^–1 NAA and 0.1 mg l^–1 kinetin (ANK). Large bulblets could only be initiated on the latter. Subsequently the bulblets of 5 mm or more in diameter were trimmed and split in half, and secondary plantlets were regenerated on MMS-medium containing ANK or MS-medium without any growth regulators which in turn grew into bulblets suitable for splitting within 12–16 weeks. A total of 700–1000 bulblets could be obtained from each initial bulb within 12 months. Anatomical studies showed that the shoots were initiated from the epidermis and hypodermis on the abaxial surface of the meristematic tissue of the basal plate of the bulb scale. This technique is useful for the multiplication and preservation of a genotype, since plantlets regenerated in this manner should be genetically uniform.