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.
Michael J. Tanabe and Nicole Wakida
Luis Humberto Escobar Torres, Eduardo Alejandro Olate Muñoz, Miguel Jordan, and Marlene Gebauer
Callus induction (CI) and later shoot induction (SI) were studied in Leucocoryne purpurea, a native and endemic Chilean geophyte species. Basal leaf portions (BL), bulb basal plate (BP), and root tips (RT) from in vitro plants were used as explants. Treatments for CI included all three explants and media containing different sources and concentrations of auxins and cytokinins as plant growth regulators (PGRs). Plant material was initiated on MS basal medium (Murashige and Skoog, 1962), supplemented with vitamins, 30 g·L-1 sucrose, 6.0 g·L-1 agar and pH adjusted to 5.7 before autoclaving. The experiments were carried on a growth chamber at 24 ± 1.5 °C. CI cultures were maintained in darkness for 16 weeks, and SI for 12 weeks in a 16-hour photoperiod. BL and RT explants did not respond to any of the CI treatments. BP explants cultured on MS basal medium without PGRs also did not produce any callus. The average frequency of callus induction for BP was 78% and the average fresh weight of callus was 10.06 g/explant after 16 weeks of culture. Best treatment for CI was BP cultured on 4.52 μm 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with 0.45 μm 6-benzyladenine (BA), when they were compared to 2,4-D alone or picloram as auxin source. After 16 weeks of culture, calli were transferred to SI medium, supplemented with three different concentrations of thidiazuron (TDZ), either intact or subdivided (150 mg/explant). SI treatments had a greater and significant response when the callus came from a CI medium containing auxin and cytokinin combined, in comparison to those coming from a CI medium containing auxins only.
Ann M. Chanon and R. Daniel Lineberger
The genus Betula contains many important forest and ornamental species and a method of rapid clonal propagation of superior genotypes is needed. Thidiazuron (TDZ) is a potent synthetic plant growth regulator with cytokinin-like activity. TDZ was used to differentiate shoots after long term exposure to dichlorophenoxyacetic acid (2.4-D) as part of a larger study on clonal fidelity. Birch calli were cultured on Woody Plant Medium supplemented with 10-5 M 2,4-D for up to 30 weeks. The calli were transferred to media containing TDZ at concentrations of 10-6 to 10-9M. Most of the tissue which had not been exposed to 2.4-D differentiated shoots five weeks after being exposed to 10-6M TDZ. Increasing the of time exposure to 2.4-D or decreasing the concentration of TDZ delayed differentiation. Calli exposed to 2.4-D for more than 18 weeks rarely differentiated shoots regardless of the concentration of TDZ used.
S. Jayasankar and U.L Yadava
Petiole discs from young leaves of female papaya (L-45) plants were cultured in MS or B5-based media containing 0, 2.25, 4.5, 11.25, and 22.5 μm 2,4-D. Compact embryogenic callus emerged from vascular tissue of petiole discs in about 3 weeks. In MS medium, 66% and 51% explants formed embryogenic callus with 11.25 and 22.5 μm 2,4-D, respectively. On the other hand, 79% explants formed embryogenic callus in B5-based medium with 4.50 μm 2,4-D. However, explants became necrotic in B5-based medium with 22.5 μm 2,4-D. Subculturing callus in auxin-free medium resulted in the development of roots or somatic embryos. Microscopic observations revealed that the roots were produced only by the callus that had retained its continuity with the vascular tissue. This investigation revealed that petioles from field grown papaya plants are potential explants for somatic embryogenesis and 2-week exposure to 2,4-D is adequate for inducing morphogenesis. Additionally, an interaction between 2,4-D and the components in the MS and B5-based media was observed.
J.T.A. Proctor, T. Slimmon, and P.K. Saxena
Ginseng is an herbaceous perennial that grows in the understorey of deciduous hardwood forests and is also cultivated for its highly valued root. The primary method of propagation of ginseng is by seed which requires the breaking of dormancy by stratification, a process which takes 18–24 months. Investigation of factors controlling the growth and development of ginseng plants is a prerequisite to the development of a more efficient system of ginseng propagation. We have recently modulated the morphogenetic potential of geranium roots and stimulated de novo development of shoots and embryo-like structures which later formed whole plants using thidiazuron (TDZ). Our objective was to investigate the morphological changes in seedling and mature ginseng plants induced by TDZ, particularly in relation to root and shoot morphogenesis and economic yield. Applications of TDZ (0.22 and 2.20 ppm), either as foliar sprays or soil watering to greenhouse-grown seedlings over 18 weeks (2 weeks after sowing to 20 weeks when plants were harvested) induced similar effects. These responses included increased stem length and diameter, and shoot and root weight (economic yield). Single foliar applications of TDZ at 62.5 and 125 ppm to 3-year-old field-grown ginseng plants 3 months before harvest increased root biomass (economic yield) by 19% to 23%. Roots of TDZ-treated seedlings and 3-year-old field-grown plants developed thickened secondary roots on the upper part of the taproot. The root-like structure of these secondary roots was confirmed by histology. In addition, TDZ treatments induced adventitious buds on the shoulder of 3-year-old roots. These buds developed into shoots to give multi-stem plants following a period of dormancy, which was overcome with GA3 (gibberellic acid) treatment before planting.
Mohamed F. Mohamed, Dermot P. Coyne, and Paul E. Read
Plant regeneration has been achieved in two common bean lines from pedicel-derived callus that was separated from the explant and maintained through successive subcultures. Callus was induced either on B5 or MS medium containing 2% sucrose and enriched with 0.5 or 1.0 mg thidiaznron/liter alone or plus various concentrations of indoleacetic acid. The presence of 0.07 or 0.14 g ascorbic acid/liter in the maintenance media prolonged the maintenance time. Up to 40 shoot primordia were observed in 4-week-old cultures obtained from 40 to 50 mg callus tissues on shoot-induction medium containing 1-mg benzyladenine/liter. These shoot primordia developed two to five excisable shoots (>0.5 cm) on medium with 0.1-mg BA/liter. A histological study confirmed the organogenic nature of regeneration from the callus tissues. The R2 line from a selected variant plant showed stable expression of increased plant height and earlier maturity. Chemical names used: ascorbic acid, N- (phenylmethyl)-1H-pnrin-6-amine [benzyl-adenine, BA], 1H-indole-3-acetic acid (IAA), N- phenyl-N'-1,2,3-thiadiazol-5-ylurea [thidiazuron, TDZ].
Guirong Li, Ran Quan, Chaohui Yan, Xiaojin Hou, and Huiling Hu
Grape (Vitis vinifera) is among the world’s most important fruit crops and is a commonly used woody plant for genomics and post-genomics research. NAC transcription factors play central roles in plant growth and development, floral organ morphogenesis, and responses to biological stress. It is therefore important to identify key transcription factors from grape and clarify their mechanisms of action to generate genetic resources for grape molecular improvement. Our research group previously cloned a NAC transcription factor from V. vinifera ‘Yatomi Rosa’ [drought and leaf roll gene 1 (DRL1)] and demonstrated that it caused dwarfing of tobacco (Nicotiana benthamiana) plants when overexpressed. In the present work, we demonstrate that overexpression of DRL1 in transgenic tobacco delays flowering time and markedly reduces pollen viability. Furthermore, crosses between male DRL1 transgenic tobacco and female wild-type tobacco exhibit substantially lower fruit set, fruit and seed weights, fruit and seed shape indices, and seed germination rates than selfed wild-type plants or crosses with a transgenic female parent. DLR1 overexpression strongly influences flowering time and reproduction in transgenic tobacco, primarily through its effects on pollen development. These results provide a foundation for further functional characterization of DLR1 in grape.
Jeffrey W. Adelberg, Bill B. Rhodes, Halina T. Skorupska, and William C. Bridges
Adventitious and axillary shoots of melon (Cucumis melo L.) were cultured from explants on a modified Murashige and Skoog medium containing 10 μm BA. Explants were diversified with regard to genetic source (breeding lines Miniloup, L-14, and B-line), seed parts (apical and cotyledon tissue), seed maturity (10-40 days after pollination; DAP), and cotyledon sections with respect to apical-radicle axis (distal and proximal). Plants were screened for ploidy level by pollen morphometry. Immature cotyledons produced more tetraploid regenerants than mature cotyledons from seed of breeding line Miniloup; the highest frequency of tetraploid regenerant plants was from cotyledons of embryos harvested 18 and 22 DAP. Explants from the apical meristem of the same seeds produced fewer or no tetraploid plants. Proximal sections from immature cotyledons of three genotypes (Miniloup, L-14, B-line) produced higher frequencies of tetraploids than whole mature cotyledons or whole immature cotyledons.
M.R. Pooler and R. Scorza
Shoots were regenerated from cotyledons of mature stored seed of three peach rootstock cultivars (`Flordaguard', `Nemared', and `Medaguard'). Shoot regeneration rates were highest when cotyledons were cultured for 3 weeks in darkness on Murashige and Skoog (MS) medium with 2.5% sucrose and a combination of IBA (1.25 or 2.5 μm) and TDZ (6.25 or 12.5 μm). Regeneration rates for `Flordaguard', `Nemared', and `Nemaguard' were as high as 60%, 33%, and 6%, respectively. Length of seed storage (1 to 3 years) did not affect regeneration rates. Seventy percent of regenerated shoots produced rooted plants. This regeneration method is rapid and simple, and stored seed can be used year-round. It may be a useful regeneration system for gene transfer in seed-propagated peach rootstocks. Chemical names used: 5 indole-3-butyric acid (IBA); thidiazuron (TDZ).
C.D. Robacker and W.L. Corley
A micropropagation system to obtain plants from inflorescences of pampas grass (Cortaderia selloana Schult. `Pumila') was developed. Factors examined included developmental stage of inflorescence cultured and growth regulator combinations and concentrations that support explant establishment, shoot regeneration, and rooting. Immature inflorescences ≈300 mm long formed many shoot primordia when initially cultured on Murashige and Skoog basal medium containing 4.5 μm 2,4-D and 8.9 μm BA and subcultured to medium with 0.4 μm 2,4-D and 4.4 μm BA. Thereafter, monthly transfer to a medium without growth regulators yielded about three shoots per tube per month for more than 6 months. Most shoots rooted spontaneously and were easily hardened to greenhouse conditions. Field-tested plants flowered within 2 years and nearly all appeared identical to the parent cultivar. With this technique, several thousand plants can be obtained from a single inflorescence in 1 year. Chemical names used: N -(phenylmethyl)-1 H -purine-6-amine (BA); (2,4-dichlorophenoxy)acetic acid (2,4-D).