To improve the efficiency of iris plant regeneration, we tested the influence of several combinations of Kin and NAA in culture media on the induction of morphogenesis and the subsequent development of plantlets. The highest rates of regeneration (67%) were consistently observed in induction media containing 0.5 μm NAA and either 2.5 or 12.5 μm Kin. Developing medium containing 1.25 μm BA was optimal for high regeneration rates and a high percentage of plantlets simultaneously developing shoots and roots. Rooted plantlets were easily acclimatized and transplanted to various soil mixtures, then grown in the greenhouse. Under optimal conditions as many as 8000 plantlets could be regenerated from 1 g of cells in ≈4 months. Chemical names used: kinetin (Kin); 1-naphthaleneacetic acid (NAA); N6-benzyladenine (BA).
Yuexin Wang, Zoran Jeknić, Richard C. Ernst, and Tony H.H. Chen
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).
Chiu-Yueh Hung and Jiahua Xie
A method of in vitro plant regeneration for both the selenium-hyperaccumulator Astragalus racemosus ‘Cream Milkvetch’ and the nonaccumulator Astragalus canadensis ‘Canadian Milkvetch’ was developed with two induction media, M1 and M2. The M1 and M2 contain Murashige and Skoog basal medium plus vitamins, 8.07 μm N-(2-chloro-4-pyridyl)-N′-phenylurea, 2.5% (w·v−1) sucrose, 0.7% (w·v−1) agar (pH 5.7), and 0.89 μm or 3.12 μm a-naphthaleneacetic acid, respectively. In vitro cultures were initiated on these two types of media with three types of explants: cotyledons, hypocotyls, and roots. More than 93% of cultured explants from both species could form calli or calli with shoots. With regard to shoot formation, A. canadensis could produce multiple shoots from all types of explants more efficiently than A. racemosus. The highest shoot induction was approximately three shoots per explant in A. racemosus, whereas A. canadensis could reach ≈10 shoots per explant. M1 could induce more shoots than M2 no matter what type of explant was used, but the overall induction rates were no significant difference. Among the three types of explants used, the cotyledons were the best explants for shoot induction in A. canadensis, whereas hypocotyls were the best in A. racemosus. In A. racemosus, shoots could also be obtained from calli on the rooting medium containing Murashige and Skoog basal plus vitamins, 2.84 μm indole-3 acetic acid, 2.5% (w·v−1) sucrose, and 0.7% (w·v−1) agar (pH 5.7). Approximately 43% of A. canadensis shoots and 19% of A. racemosus shoots could be rooted on the rooting medium.
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).
Ana Carolina Espinosa, Paula M. Pijut, and Charles H. Michler
A complete regeneration protocol was developed for Prunus serotina Ehrh., an important hardwood species for timber and sawlog production in the central and eastern United States. Nodal sections were cultured on Murashige and Skoog (MS) medium supplemented with 4.44 μm 6-benzylaminopurine (BA), 0.49 μm indole-3-butyric acid (IBA), and 0.29 μm gibberellic acid (GA3). In vitro leaf explants of three genotypes were placed on woody plant medium (WPM) supplemented with 0, 2.27, 4.54, or 6.81 μm thidiazuron (TDZ) in combination with 0, 0.54, 1.07, or 5.37 μm naphthaleneacetic acid (NAA), and on WPM supplemented with 0, 4.44, 8.88, or 13.32 μm BA in combination with 0, 0.54, 1.07, or 5.37 μm NAA. Cultures were maintained either in continuous darkness for 5 weeks, or in the dark for 3 weeks and then transferred to a 16-hour photoperiod. TDZ and the genotype had a significant effect on the number of shoots regenerated. The maximum mean number of shoots regenerated per explant (5.05 ± 1.14) was obtained with 2.27 μm TDZ plus 0.54 μm NAA with the 3-week dark period then light treatment. The highest percent shoot regeneration (38.3) and mean number of shoots (4.13 ± 0.97) was obtained with 6.81 μm TDZ plus 1.07 μm NAA. The highest rooting (27%) of adventitious shoots and number of roots per shoot (2.3 ± 0.2) was obtained with 2.5 μm IBA when shoots were maintained for 7 days in the dark on rooting medium before transfer to a 16-hour photoperiod. The highest rooting (70%) of nodal explant-derived stock cultures and number of roots per shoot (2.7 ± 0.9) was also obtained with 2.5 μm IBA, but when shoots were maintained for 4 days in the dark before transfer to a 16-hour photoperiod. In total, 86% of the plantlets survived acclimatization to the greenhouse and 100% survival after overwintering in cold-storage.
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.
I. Arrillaga and S.A. Merkle
A protocol to achieve efficient plant regeneration from juvenile black locust (Robinia pseudoacacia L.) explants is described. Direct adventitious shoots were induced from cotyledon explants on woody plant medium containing 22.2 μm BA and 0.4 μm 2,4-D. Shoots developed and new shoots were induced when the explants were transferred to medium without growth regulators. The effect of dark incubation on shoot regeneration from cotyledons indicated that 15 days of darkness resulted in a high regeneration frequency (91.7%). Adventitious shoot formation also was induced from sections of in vitro-derived leaves cultured in darkness on Murashige and Skoog medium supplemented with 4.4 μm BA and 24.6 μm IBA. A shoot regeneration frequency of 89% was obtained when explants were subcultured on a medium containing 4.4 μm BA and 0.5 μm IAA. Shoots were rooted on Schenk and Hildebrandt medium with or without IBA. Plantlets were acclimatized and grown in the greenhouse. Chemical names used: N -(phenylmethyl)-1H -purin-6-amine (BA); 2,4-dichlorophenoxyacetic acid (2,4-D); indole-3-acetic acid (IAA); indole-3-butyric acid (IBA).
Enio Tiago de Oliveira, Otto Jesu Crocomo, Tatiana Bistaco Farinha, and Luiz Antônio Gallo
A protocol for large-scale Aloe vera (L.) Burm. f. production was established using micropropagation of apical buds. The effects of two chlorine-based disinfectants were evaluated on the survival of the explants in different treatments in a semisolidified Murashige and Skoog (MS) medium in the presence of 6-benzylaminopurine (6-BAP; 2 mg·L−1). During 120 days, 136 green apical shoots bearing axillary buds were multiplied four times at 30-day intervals in the same MS medium, reinoculating seven to nine explants per flask each time. The elongation and rooting processes were carried out in the same MS medium without 6-BAP. A total of 40,495 Aloe vera microplants were obtained, a yield of 300 microplants per apical bud at a rate of 1:5.3 in every multiplication period of 30 days. From that total, 38,480 Aloe vera microplants were successfully acclimatized transferring to 36- and 64-cell polyethylene trays containing proper substrate in two different ex vitro greenhouse conditions. After a 3-month period, fresh and dry matter weights of the Aloe vera plants were determined. All the data from each experimental phase were statistically analyzed. The use of 64-cell (40 cm3/cell) trays represented an economy of 47.37% in greenhouse space and 50% in the amount of substrate per Aloe vera plant.
Mohamed F. Mohamed, Paul E. Read, and Dermot P. Coyne
Dry seeds from two lines of common bean (Phaseolus vulgaris L.) and one cultivar of faba bean (Vicia faba L.) were germinated on Murashige and Skoog (MS) medium containing B vitamins, 30 g sucrose/liter, and either 2.5, 5.0, or 7.5 μm benzyladenine (BA). Axenic seed cultures were grown at 22 to 24C in darkness and under continuous light from cool-white fluorescent tubes (40 μmol·m-2·s-1). Explant tissues were prepared from cotyledonary nodes (CN) and primary nodes (PN) of 14-day-old seedlings. Explants were cultured on corresponding seedling growth medium and maintained under continuous cool-white light (40 μmol·m-2·s-1). The percentages of CN and PN (in one line of common bean) explants that regenerated shoots and the number of shoots per explant (in all germplasm) were highest when nodal tissues were prepared from seedlings germinated in darkness. These responses were optimal on medium containing 5 μm BA during seedling growth and subsequent culture of explants. The number of shoots per explant was two to five times higher on explants cultured on medium with 0.25 to 1.0 μm forchlorfenuron (CPPU) or thidiazuron (TDZ) than on medium with 5 μm BA. Higher (2.5 and 5 μm) CPPU and TDZ concentrations inhibited shoot elongation and stimulated callus production. Histological analyses indicated that adventitious meristems formed 6 to 8 days after explant culture. Progenies from regenerated plants appeared similar to plants raised from the original seed stocks. Chemical names used: N- (phenylmethyl) -1 H- purin-6-amine (benzyladenine, BA); N- (2-chloro-4-pyridyl)-N'- phenylurea (forchlorfenuron, CPPU); N- phenyl -N' -1,2,3-thiadiazol-5-ylurea (thidiazuron, TDZ).
Gerson R. de L. Fortes and Silvio L. Teixeira
The aim of this work was to study different apple of somatic material as callus and adventitious shoots are concerned, for further utilization in the research of somaclonal variation. The somatic materials were: leaf discs, cotyledons and hypocotyls of Gala apple seedlings, cultivated in a MS medium added by B5 vitamins in addition to (in mg/l): BAP (1,0), NAA (0,5) mio-inositol (100,0) sucrose (30,0 g/l) and solidified in agar (6,0 g/l). The several times of explant exposition to the dark affected the final callus weight. Callus weight derived from leaf discs were higher than those for cotyledons and hypocotyls. Explants exposed directly under light or up to two weeks in the dark showed less percentage of regenerative callus as compared to those of three weeks in the dark. The leaf explants presented the highest percentage of regenerative callus. The least response was obtained for those derived from hypocotyls. The highest number of adventitious shoots was obtained keeping the explants three weeks in the dark as compared to directed light exposition.