Fast growing callus was derived from Cephalotaxus harringtonia stem explants placed on MS basal salt medium with B-5 vitamins, 3% sucrose, 1 μM kinetin and 4.5 μM 2,4-D. Callus placed on basal medium with 10 μM kinetin and 0.45 μM 2,4-D turned green and organogenesis was observed upon subculture onto basal medium without hormones. Shoots were excised and placed on 1/2 strength MS salts and 10% sucrose for further shoot development. During the process of organogenesis, we also observed the differentiation of roots. Rapidly growing root cultures were established by culturing them under a 24 hour light regime of 35 μM/m2/s. Two grams of root tip explants cultured on B-5 medium with 2% sucrose were capable of producing an average of 24 grams of roots within 11 days. A 20-fold increase in fresh weight was achieved within 3 weeks when 15 grams of these roots were cultured in a 3 liter air-sparged bioreactor. C. harringtonia contains a number of alkaloids that exhibit cytotoxicity and are being evaluated as chemotherapeutic agents. We are currently in the process of establishing growth characteristics for these roots and assaying roots for the presence of these alkaloids. All cultures were grown under a 12 hour light regime unless otherwise stated.
Enaksha R. Wickremesinhe and Richard N. Arteca
Chang-Yeon Yu and John Masiunas
Repeated callus sub-culture reduce the regeneration capacity in many species. Our studies determined the effect of genotype and medium on regeneration of several Solanum and Lycopersicon genotypes from long-term callus cultures. In the first study, 13 genotypes were transferred to regeneration medium, including: Murashige and Skoog plus Gamborg Vitamins (MG); Murashige and Skoog (MS); Gamborg (GM); and white (WM). The greatest shoot regeneration was on the MG medium, containing the highest levels of thiamine. Shoot differentiation was greatest with 0.2 mg/l IAA and 2 mg/l BA. No plants were regenerated on GM or WM medium. In a second study, the effect of thiamine (0 to 200 mg/l) on shoot regeneration of the L. peruvianum genotypes PI199380, PI126945, PI251301, and PI128652, along with Solanum ptycanthum, Solanum nigrum, and L. esculentum `Diego' was evaluated. Shoot regeneration of Solanum ptycanthum, Solanum nigrum, L. peruvianum PI 199380 and PI25301 was best with 20 mg/l of thiamine.
Karim H. Al-Juboory, J. Al-Naimi, L.K. Al-Amiry, R. Shibli, and R.M. Skirvin
Callus was initiated from leaves of Gladiolus cv. `Balady' on MS medium containing 1.0 mg/L NAA, 0.1 mg/L 2,4-D, and 0.5 mg/L kinetin. Organogenesis from callus was induced on medium containing 0.5, 1.0, 1.5, or 2.0 mg/L of either BA, kinetin, or TDZ. TDZ was more effective and resulted in a higher percentage regeneration and regenerant number. The microshoots produced were then propagated in vitro and cormel production was studied. Maximum shoot number (25.1) was obtained on medium containing 1.0 mg/L TDZ without auxin supplements in liquid shaking culture. In vitro cormel formation was significantly enhanced by B-9 and paclobutrazol. Increased sucrose concentration (4% to 5%) proved the most effective for cormel formation. Optimal dormancy break was obtained by storing cormels at 5°C for 1 month or by soaking them for 5 sec with 50 mg/L GA3. In-vitro rooting was achieved on solid medium containing NAA, IAA, or IBA, with higher root number recorded on NAA-treated cultures. Rooted microshoots were successfully acclimatized for ex vitro conditions and grown in the greenhouse. Plants produced from in-vitro propagation showed similar morphological characteristics of plants propagated by direct corm planting in the greenhouse.
Jeffrey D. Griffin and Margaret S. Dibble
Genetic transformation of Kentucky bluegrass (Poa pratensis L.) requires methods for high-frequency regeneration of plantlets from cultured cells. Regeneration of this important turf species has been reported, albeit at a low frequency from seed-derived callus. We tested the potential of 3 synthetic auxins, used in the callus initiation and growth medium, for their ability to promote regeneration in 3 bluegrass varieties. 10 μM 2,4-D promoted regeneration from 0 to 5% of calli, 30 μM and 60 μM picloram promoted regeneration from 0 to 8% of calli, and 10 to 60 μM dicamba, in combination with BA, promoted regeneration from 1% to 8% of calli. In a subsequent experiment, both 60μM and 90 μM dicamba, with 20 μM BA, promoted regeneration from 45% of calli averaged across varieties. These media were tested for the promotion of regeneration in 12 diverse bluegrass varieties. Although up to 45% of the calli from some varieties regenerated plantlets, the response of other varieties was markedly lower, indicating a genetic component in the response to these media.
Michael J. Tanabe and Nicole Wakida
Noni, Morinda citrifolia, is receiving a lot of attention for its potential medicinal effects. Hawaii is an ideal growing environment for this plant, where it has been used for many purposes, including medicinal ones, by ancient Polynesians. Currently, there is a rapidly developing noni industry in the state of Hawaii. Propagation of this plant is almost exclusively by seeds, and germination generally requires a couple of months without preconditioning or about a month if mechanically scarified. We developed an in vitro protocol that significantly improves percent germination rate by altering incubation temperature and the in vitro culture basal medium. Germination time was decreased to 4 days when the embryo was extracted and exposed to 31 °C. A basal medium containing 1/2 Murashige and Skoog (M&S) salts was the most effective in reducing germination time and increasing percent germination. Stem pieces obtained from in vitro-propagated seedlings produced callus when explanted in 1/2 M&S containing various levels of naphthalene acetic acid (NAA). The most effective treatment was 0.5 μm NAA and the least effective treatment was 2 μm NAA. Treatments without NAA did not produce callus. Calli treated with 4.40 μm 6-benzylaminopurine (BA) or 8.80 μm BA were the most effective in promoting caulogenesis. We also demonstrated that the number of first generation seedlings produced from each embryo could be increased by treatment with 8.80 μm BA.
Takuma Ishizaki, Fuminori Komai, Kiyoshi Msuda, and Chiaki Megumi
Effects of ethephon, AgNO3, and AVG on formation of embryogenic callus and somatic embryogenesis were examined in cultures of explants of spinach (Spinacia oleracea L. `Nippon') roots. At 10 μm, ethephon more than doubled the frequency of formation of embryogenic callus as compared to that in cultures without ethephon. Silver nitrate at 10 mm and AVG at 1 μm each inhibited formation of embryogenic callus but neither reduced the growth rate of established callus. When ethephon was applied at 10 μm during embryogenesis, it completely inhibited embryo development. By contrast, AgNO3 at 10 μm markedly increased the number of embryos. Results suggest that ethylene might be essential as a plant hormone for formation of embryogenic callus but inhibits somatic embryogenesis per se in cultures of explants of spinach roots. Chemical names used: 2-chloroethylphosphonic acid (ethephon), silver nitrate (AgNO3), aminoethoxyvinylglycine (AVG).
T. Gregory McCollum, Hamed Doostdar, Michael Burkhart, Randall Neidz, Richard T. Mayer, and Roy E. McDonald
Inhibition of the growth of fungal pathogens has been related to levels of a β-1,3-endoglucanase (EC 184.108.40.206) (GLU) in citrus as well as other plant species. Our long-term objective is to transform Citrus spp. to express enhanced levels of GLU with the aim of increasing resistance to fungal pathogens. We have purified a β-1,3-endoglucanase from nonembryogenic Citrus sinensis (L.) Osbeck cv. Valencia callus to electrophoretic homogeneity by means of pH precipitation and ion exchange chromatography. The protein has an apparent M r of 32.5 and a pI > 10. The enzyme hydrolyzes laminarin (Laminaria digitata) optimally at pH 5 and 50°C. The enzyme will hydrolyze any glucan polymer with a β-1,3 linkage whether soluble or insoluble and the rate of hydrolysis is proportional to the relative abundance of β-1,3 linkages. The enzyme does not hydrolyze cellulose or starch. Product characterization by thin-layer chromatography indicates that the enzyme is an endohydrolase. Initial attempts to sequence the protein indicated that it was N-terminally blocked. Therefore the protein was hydrolyzed using AspN, the fragments separated by SDS-PAGE, blotted onto nitrocellulose, and one of the fragments was sequenced. Amino acid sequence analysis indicated that the protein shared homology with a number of β-1,3-endoglucanases. Antibody to the purified protein was raised in rabbits and used to screen an amplified cDNA library prepared from Citrus sinensis (L.) Osbeck cv. Valencia callus. One of the positive clones was selected and sequence analysis indicated that the clone was homologous with other β-1,3-endoglucanases.
L. Chen, S.O. Park, S. Dhir, and A.S. Bhagsari
Several experiments were conducted to evaluate the influence of explant type, sucrose level, and callus development time on sweetpotato [Ipomoea batatas (L.) Lam] in vitro culture. Shoot tip, petiole, and leaf of Selection 75-96-1 was used as explants in Murashige and Skoog (MS) media with different plant growth regulators. Calli derived from shoot tip and petiole produced 42.1% and 10.3% somatic embryos, respectively, but the leaf failed to produce somatic embryos. The effect of sucrose level was determined using shoot tip as explants. Compared with 3% sucrose in the same plant growth regulators level medium during callus initiation and callus proliferation periods, 5% sucrose level suppressed root growth and improved shoot regeneration. The callus development time was measured by using shoot tips on callus initiation medium containing 1.5 mg/L alpha-Naphthaleneacetic acid (NAA) and 0.25 mg/L Kinetin (KIN) plus 5% sucrose. When explants were cultured for less than 6 weeks during callus initiation, then transferred onto plant regeneration medium, plant regeneration via organogenesis occurred; whereas, maintaining cultures for more than 12 weeks on the same callus initiation/proliferation medium, plant regeneration was favored via embryogenesis. Explant type and other factors affecting plant regeneration noted here could be applied to protoplast culture, somatic hybridization, and transformation in sweetpotato.
Norman E. Pellett and David A. Heleba
Most of 36 crabapple and 19 other woody plant taxa demonstrated the ability, when dormant, to grow a continuous row of callus along the cambial region on split-stem pieces within 5 to 7 days of incubation at 25 °C. The ability to grow callus after freezing tests was compared with discoloration and electrical conductivity for determining laboratory freeze injury to selected taxa. Hardiness levels were determined using the procedures of callus growth, discoloration, and electrical conductivity after freezing stem pieces of Jack crabapple [Malus baccata (L.) Borkh. `Jacki'], pink bud Sargent crabapple [M. sargentii Rehd. `Rosea'], Mary Potter crabapple [Malus sp. `Mary Potter'], and snowberry mountainash [Sorbus discolor (Maxim.) Maxim.]. Sampling dates for laboratory freezing tests were chosen to represent midwinter cold hardiness and partial hardiness of either late fall or early spring. There was a high correlation between discoloration and callus ratings for most plants; however, the two methods usually did not identify the same critical temperature (T50) for injury. The critical temperatures identified by callus growth was often 3 to 6 °C lower than for discoloration. For many taxa, callus growth was easier to see than discoloration of cambium and phloem, providing a less subjective evaluation of injury. TTC (2,3,5-triphenyl tetrazolium chloride) treatment was sometimes useful to identify callus growth that died after forming. The critical temperature (Tc), the highest temperature at which relative electrical conductivity differed significantly from the control temperature, was higher in most cases, indicating less cold hardiness than the T50 for callus and discoloration. The callus procedure may have value for evaluating injury to the cambial zone from freezing and other plant stresses because it determines the ability of the plant to continue growth.
Aaron Brown and Harrison G. Hughes
Callus induction and regeneration of alkaligrass (Puccinellia distans) was developed in our laboratory for use in transformation studies of turfgrass. Particle bombardment of the embryogenic callus is being evaluated using a helium particle inflow gun constructed at Colorado State Univ., according to the design of Philippe et al. (Ohio State Univ., 1993). Its utility in delivering DNA to plant cells is being tested by measuring the frequency of transient gene expression of a reporter gene (GUS pBI121) in embryogenic callus of alkaligrass. Varying pressure of helium and the distance of the calli in the chamber are also being evaluated for efficiency in transformation.