Guava (Psidium guajava L.), also called `apple of tropics,' is immensely nutraceutical and horticulturally important. Being a tropical plant, it cannot stand temperatures below 25° F and needs frost protection to grow in temperate regions. To adapt in cold climate, cold hardy guava cultivars are needed. Conventional ways are uneconomic in time and efforts. Still, transgenic plants developed using biotechnological approaches of tissue culture and rDNA technology, appear to have great potential. Thus, protocols for in vitro propagation of guava were developed via organogenesis and somatic embryogenesis using nodal explants from mature trees and young zygotic embryos, respectively. Nodal explants induced multiple shoots when cultured on MS medium fortified with KIN, BAP and Ad.S. Adding a (NO3)2 to medium was useful to prevent in vitro shoot tip browning of adventitious shoots. Rocker liquid culture greatly increased growth of multiple shoots compared to the agar-based medium. It appears to be a good tool for woody plant tissue culture. Induction of somatic embryos in guava was also achieved on MS medium supplemented with IAA auxin. About 80% to 90% somatic embryos germinated normally. To achieve Agro-bacterium-mediated gene transfer in guava, on-going co-cultivation of organogenic tissues of guava is to optimize protocols for freeze tolerance gene (CBF1, CBF2, CBF3) transfer. Plasmid vectors containing selectable markers (nptII gene for antibiotic selection and GUS reporter gene as scorable gene mediated selection), with CaMV 35S promoter gene has been introduced into guava tissues and the resultant plants showed antibiotic resistance. Details of the experimental procedures and up-to-date results will be discussed.
Bipul K. Biswas*, Nirmal Joshee, and Anand K. Yadav
Mohamed F. Mohamed, P. E. Read, and D. P. Coyne
Regeneration in vitro from the embryonic axis in Phaseolus sp. has not been reported. Two embryo sizes, 0.3-0.4 mm and 0.6-0.7 mm long at 10-12 and 21 days after pollination, respectively, were excised from 4 P. vulgaris (P.v.) and 2 P. acutifolius (P.a.) genotypes. The embryonic leaves and radicale were removed, and 0.1-0.2 mm of the embryonic axis was cultured on Gamborg's B5 medium with 0, 5, 10 and 20μ MBA. The cultures were incubated in the dark at 25°C for 2 weeks followed by 1 week in continuous cool white light (25μ MS-1m2) before transferring to the second medium (0, 2μ MBA and 2μ MBA + 4μ MGA3). The tissues from the larger embryos initiated a single shoot without PGR in 30% of 1 P.v. explants and 30-60% in 2 P.a. The other 3 P.v. formed roots only. Multiple shoots were initiated in all P.v. (15-60%) and in 2 P.a. (60 and 70%) with 5 or 10μ MBA. The tissues from the smaller embryos had single shoots for all genotypes (30-60%) without PGR. Multiple shoots were initiated in 50-80% and 75-90% of the explants from P.v. and P.a., respectively, with 5 or 10μ MBA. Excess callus formed with 20μ MBA and regeneration decreased. After 3 weeks on the second medium, 6-8 shoot s/P. v. and up to 15-20 shoots/Pa. explants were observed.
Young Goel Shon, Joong Choon Park, and Byoung Ryong Jeong
Effect of combination and concentration of growth regulators on the regeneration of pepper plant from different explant tissues was studied. Seedlings were grown aseptically in 400 ml glass bottles containing MS agar medium at 26±2C under a 16 h·d-1 photoperiod (2000 lux, florescent lamps). Explants taken from 4 week-old seedlings were cultured under these conditions on 40 ml of MS agar (8 g·liter-1) medium containing 3 g·liter-1 sucrose in a 400 ml glass bottle. Primary and subsequent leaves attached to petiole regenerated better than cotyledon and hypocotyl. Among the combinations of different concentrations of cytokinin and auxin added in the medium, a combination of 5 μM IAA with either 10 μM zeatin or 10 μM BA gave the best regeneration. With these combinations, regeneration frequency of multiple shoots from the primary and subsequent leaves was greater than 70%. Regenerated shoots rooted readily in MS agar medium containing 3 g·liter-1 sucrose and 0.5 μM NAA.
Yan Ma, David H. Byrne, Jing Chen, and Amanda Byrne
Several rose species (Rosa rugosa, R. wichuraiana, R. setigera, R. laevigata, R. banksiae, R. roxburghii, R. odorata and hybrids) were employed to establish the appropriate nutrient media for shoot multiplication and root initiation of cultured shoots and to describe a procedure for the successful transfer to soil of plants obtained in vitro. Cultured shoot tips and lateral buds from different genotypes proliferated multiple shoots on a basal medium (MS salt, vitamins, glycine, sucrose and agar) supplemented with 0mg/l to 6mg/l 6-benzylamino purine (BA) and 0mg/l to 0.5 mg/l naphthalene acetic acid (NAA). Most rose species cultured in a modified MS medium supplemented with 2mg/l BA showed good growth and shoot proliferation. The buds nearest the apex exhibited the slowest rate of bud development. Root development was enhanced and shoot development inhibited by lowering the concentration of MS salts to quarter- and half-strength. With difficult-to-root species, rooting was improved by supplementing the media with auxin or giving them 3-7days of dark treatment.
Eric W. Mercure, Carol A. Auer, and Mark H. Brand
Tissue proliferation (TP) is characterized primarily by the formation of galls or tumors at the crown of container-grown rhododendrons propagated in vitro. However, TP of Rhododendron `Montego' is observed initially in in vitro shoot cultures and it is characterized by the formation of multiple shoots with small leaves and nodal tumors. The formation of shoots in `Montego' TP (TP+) shoot cultures occurs without the presence of exogenous cytokinin in the medium, unlike normal `Montego' (TP–) shoot cultures, which require cytokinin for shoot growth. Structural studies have shown that tumors are composed of many adventitious buds and parenchyma cells, suggesting that TP is a result of abnormal cytokinin regulation that is controlling tumor and shoot formation. Two approaches are being used to determine if differences in cytokinin concentration and/or metabolism exist between TP+ and TP– shoot cultures. In the first approach, shoot cultures are grown in vitro for 1 week in the presence of tritiated isopentenyladenine (iP). Cytokinin uptake and metabolism are analyzed using HPLC and other analytical methods. Experiments suggest that extensive degradation and N-glucoside conjugation occur in TP+ and TP– shoots, resulting in the removal of most of the exogenous iP. In the second approach, the levels of endogenous cytokinins such as iP, isopentenyladenosine, zeatin, and zeatin riboside, are being measured in TP+ tumors and shoots and in TP– shoots by an ELISA method.
A. Raymond Miller and Craig K. Chandler
A protocol was developed for excising and culturing cotyledon explants from mature achenes of strawberry (Fragaria × ananassa Duch.). Cotyledon explants formed callus with multiple shoot buds on agar-solidified Murashige and Skoog media containing several combinations of hormones (1 μm 2,4-D; 10 μm 2,4-D; 1 μm BA + 1 μm 2,4-D; 1 μm BA + 10 μm 2,4-D; 5 μm BA; 5 μm BA + 1 μm 2,4-D; 5 μm BA + 10 μ m 2,4-D; 5 μ m BA + 5 μm NAA; 5 μ m BA + 15 μ m NAA). After three subcultures, only tissues maintained on the medium containing 5 μm BA + 5 μm NAA continued to form shoots. Tissues transferred to other media eventually died (1 μm 2,4-D; 1 μ m BA + 10 μ m 2,4-D; 5 μ m BA; 5 μ m BA + 1 μ m 2,4-D), became unorganized (1 μm BA + 1 μm 2,4-D; 5 μm BA + 10 μm 2,4-D; 5 μm BA + 15 μm NAA), or formed roots (10 μm 2,4-D). Whole plantlets were produced by transferring callus with buds to medium lacking hormones. The rapid regeneration of clonal plantlets from cotyledon explants may be useful for reducing variability in future developmental studies. Chemical names used: N-(phenylmethyl)-1H-purin-6-amine (BA); (2,4-dichlorophenoxy) acetic acid (2,4-D); and 1-naphthaleneacetic acid (NAA).
Sherry Kitto and Jeanne Frett
Hexastylis shuttleworthii is a highly ornamental shade-tolerant evergreen herbaceous plant native to the southeastern U.S. that is difficult to propagate using traditional methods. Micropropagation would make possible the wider distribution of selected clones. Seeds were surface-sterilized and germinated in vitro. Seedling clones were maintained on a MS basal medium containing 1 mg/L BA and were subcultured monthly. Proliferation of clones 2 and 3, maintained on media supplemented with 1, 2.5 or 5 mg/L BA for 6 months, increased slightly with increasing BA concentration; however, proliferation decreased slightly over the experimental period. Rooting medium (perlite, vermiculite, MetroMix 510, Bacto Growers Mix) did not effect microcutting root production or subsequent plant survival. Microcuttings rooted in vitro (67% survival) generated more leaves compared to microcuttings rooted under humidity domes with mist in the greenhouse (8% survival). After rooting in vitro, multiple-shoot clumps (95%) survived better than individual shoots (29%) under greenhouse conditions. Plants were easily established when planted in raised beds in a lath house.
Leigh E. Towill and Philip L. Forsline
The dormant vegetative bud method for cryopreservation has been successfully applied to many lines of apple. We examined this method for five cultivars (Kentish, Montmorency, Meteor, North Star, Schatten Morelle) of sour cherry (Prunus cerasus L.) with the aim of developing long-term storage at NSSL. Singlebud nodal sections (35 cm) were desiccated to 25%, 30%, or 35% moisture before cooling at 1°C/hour to –30°C and holding for 24 hours. Sections were then directly placed in storage in the vapor phase above liquid nitrogen (about – 160°C). Warmed samples were rehydrated and patch budded at Geneva to assess viability. Sections that were either undried, dried but unfrozen, or dried and cooled to –30°C survived very well. For samples then cooled to –160°C, highest viabilities for each line occurred with the 25% moisture level, although fairly high viabilities also were observed at 30% and 35% moistures. Cryopreserved buds from four lines directly developed into a single shoot; buds from Montmorency formed a shoot from a lateral within the bud, suggesting that the terminal meristem died but that axillary meristems within the bud survived and formed a shoot or multiple shoots. Nineteen lines were harvested in January 1996 for long term storage of sour cherry germplasm under cryogenic conditions.
María Luisa Osorio-Rosales and Martín Mata-Rosas
Experiments were conducted to establish an efficient protocol of micropropagation of Beaucarnea gracilis and B. recurvata two endemic and endangered Mexican species. Multiple shoots were induced by direct organogenesis from in vitro seedlings and longitudinal sections of seedlings in both species. The highest formation of shoots per explant, both B. gracilis and B. recurvata, was obtained from longitudinal sections of seedlings on Murashige and Skoog (MS) medium supplemented with 22.2 μm 6-benzylaminopurine, induced 8.2 and 11.1 shoots per explant respectively. In vitro rooting was readily achieved on MS medium with 1 g/l activated charcoal without growth regulators. According to initial treatment and depending on where the shoots come from, the rooting rates were 61% to 100% for B. gracilis, and 83% to 100% for B. recurvata. Survival rates in greenhouse conditions for both species were 80% to 100% after 3 months. These results indicate that the micropropagation of these species of Beaucarnea is technically feasible, and that in vitro culture is a useful option for the conservation and propagation of these important endangered species.
Hak-Tae Lim, Haeng-Soon Lee, and Tage Eriksson
Plant regeneration ability of ginseng (Panax ginseng) via organogenesis was studied morphologically and anatomically. Compact callus was introduced from four different types of explants—leaf, petiole, flower stalk, and root—of in vitro-grown plantlets. Petioles were found to be the best material for callus induction. Calli induced on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (1.0 mg·L–1) and kinetin (0.1 mg·L–1) were conditioned for 2 weeks on the same medium. These calli differentiated into adventitious shoots when cultured on half-strength MS basal medium plus kinetin at 1.0 mg·L–1 and STS at 2.5 mg·L–1. An addition of GA3 (1.0 mg·L–1) and BA (1.0 mg·L–1) to MS basal medium, however, induced high-frequency in vitro flowering (86.1%) and multiple shoot budding, which affected the normal, complete development of plantlets. Plantlets with well-developed root systems were obtained 6 weeks after regenerated shoots had been transplanted to half-strength MS20 medium containing IBA at 0.25 mg·L–1. Nuclear DNA content was measured to check the stability of their ploidy level. Based on DNA flow cytometric analysis, all of the regenerants were typically diploids as were the mothers plants, indicating that nuclear DNA content remained stable during cell differentiation.