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S.I. Shibairo, M.K. Upadhyaya, and P.M.A. Toivonen

The effect of potassium (K) nutrition on the shelf life of carrots was studied using a hydroponics system involving rockwool slabs as support. Carrots were grown for 192 days under greenhouse conditions and supplied with 0, 0.1, 1.0, 10, and 15 mm of K. Increase in K concentration in the nutrient medium decreased postharvest weight loss. Carrot weight and tissue K content increased and water potential, osmotic potential, and relative solute leakage decreased with increasing K concentration in the nutrient feed. Differences in postharvest weight loss were mainly associated to root weight and relative solute leakage. Root weight correlated negatively and relative solute leakage correlated positively to water loss. Water and osmotic potential also correlated to water loss, but not as strongly as root weight and relative solute leakage. These results suggest that K nutrition influences postharvest weight loss by influencing carrot size and membrane integrity. Effects on cell water and osmotic potential are also important in this regard but to a lesser extent.

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K.A. Malik, Christena Visser, and praveen K. saxena

In vitro regeneration by shoot organogenesis and-or somatic embryogenesis is accomplished by culturing the explants on a nutrient medium supplemented with phytohormones. Auxins in general, and 2,4-D in particular, have been shown to induce somatic embryogenesis whereas shoot regeneration is stimulated by cytokinins. In studying the morphoregulatory role of thidiazuron (TDZ) - a substituted urea with cytokinin-like activity - we found that it induces a high frequency of both organogenesis and somatic embryogenesis depending upon the plant species. For instance, whole seedlings of peanut developed somatic embryos and those of bean and pea produced shoots in response to culture on TDZ (1-40 μM)-supplemented media. In cultured explants of geranium, the use of TDZ (0.2-1 μM) effectively replaced the requirement of 2,4-D or BAP and IAA for obtaining somatic embryos. The frequency of regeneration was two to ten times higher than that achieved with auxin-cytokinin combinations. While no direct evidence is currently available to establish a relationship between TDZ and endogenous phytohormones, our results suggest that it may act by establishing endogenously the auxin:cytokinin ratio permissive of induction and expression of morphogenically competent cells.

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Brent Tisserat

Comparative tests were conducted to determine the influence of the culture vessel size and medium volume on the growth rates of shoot tips of peas (Pisum sativum cv. `Wando'), lettuce (Lactuca salvia) Kidney beans (Phaseolus vulgaris). Culture vessels employed included: culture tubes, baby food jars, Magenta rectangular containers, 1 -pint Mason jars, 1 -quart Mason jars, l-quart Mason jars employed with an automated plant culture system (APCS), 1/2-gallon Mason jars with an APCS, BioSafe containers with an APCS, and mega-culture chambers with an APCS. The APCS consisted of a peristaltic pump, media reservoir containing 1 liter of nutrient medium, and a culture chamber (<925 mm3). High positive correlations occurred comparing culture weight, leaf length and plant height with culture chamber volume, media volume and culture chamber height. APCSs consistently gave higher growth rates and exhibition of mature morphogenetic responses such as flowering and fruiting than growing plants on agar culture systems. Cost analysis comparing APCSs and conventional tissue culture systems is presented.

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D. Marshall Porterfield, Mary E. Musgrave, and Thomas W. Dreschel

A ground-based comparison of plant nutrient delivery systems that have been developed for microgravity application was conducted for dwarf wheat (Triticum aestivum L. `Yecora Rojo') and rapid-cycling brassica (Brassica rapa L. CrGC#1-33) plants. These experiments offer insight into nutrient and oxygen delivery concerns for greenhouse crop production systems. The experiments were completed over a 12-day period to simulate a typical space shuttle-based spaceflight experiment. The plant materials, grown either using the porous-tube nutrient delivery system, the phenolic foam support system, or a solidified agar nutrient medium, were compared by plant-growth analysis, root zone morphological measurements, elemental composition analysis, and alcohol dehydrogenase enzyme activity assay. The results of these analyses indicate that the porous tube plant nutrient delivery and the phenolic foam systems maintain plant growth at a higher level than the solidified agar gel medium system. Root zone oxygenation problems associated with the agar system were manifested through biochemical and morphological responses. The porous tube nutrient delivery system outperformed the other two systems on the basis of plant growth analysis parameters and physiological indicators of root zone aeration. This information is applicable to the current crop production techniques used in greenhouse-controlled environments.

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Ramsey Sealy, Michael R. Evans, and Craig Rothrock

Growth of Pythium aphanidermatum, Pythium ultimum, Pythium irregulare, Phytophthora nicoctianae, Phytophthora cinnomomi, Fusarium oxysporum, Rhizoctonia solani and Thielaviopsis basicoli was inhibited in vitro when grown in a clarified V-8 nutrient solution containing 10% garlic extract. After exposure to 10% garlic extract for 3 days, all fungi and fungal-like organisms failed to grow after being washed and transferred to fresh cornmeal agar nutrient medium without garlic extract. When Sphagnum peat was inoculated with P. aphanidermatum and drenched with solutions containing varying concentrations of garlic extract, a single drench of 35% garlic extract or two drenches of 15% garlic extract were required to rid the substrate of viable P. aphanidermatum. In sand, a single application of 25% garlic extract or two applications of 10% garlic extract were required to rid the sand of viable P. aphanidermatum Thus, Sphagnum peat appeared to partially inactivate the components in garlic and did so to a greater extent than sand. Therefore, efficacy of garlic extract as a soil drench fungicide will be affected by the type of substrate or soil to which the garlic extract is applied.

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Ramana M. Gosukonda, C.S. Prakash, and Ananta Porobo Dessai

Studies were conducted to improve adventitious shoot regeneration in sweetpotato [Ipomoea batatas (L.) Lam.], specifically to extend the protocol to many genotypes and to elicit production of multiple shoots per explant. The use of a two-stage procedure where excised petioles were incubated on Murashige and Skoog (MS) (1962) medium with 2,4-D (0.2 mg·liter–1) for 3 days and transferred to a second medium containing MS salts with thidiazuron and 2iP (0.05 mg·liter–1 each) resulted in shoot regeneration from eight of 13 genotypes tested, including elite sweetpotato cultivars such as `Jewel' and `Rojoblanco'. PI 318846-3 was the most regenerable genotype, with up to 77% of explants producing one to three shoots per explant. The orientation of the petiole on the nutrient medium was critical; those placed vertically inverted developed multiple shoots. Wounding explants through epidermal peeling with normal horizontal orientation of the explants during incubation also resulted in multiple shoot production (about three shoots per explant). Interference with auxin transport due to explant inversion or wounding may have stimulated increased shoot induction. Chemical names used: 2,4 dichlorophenoxyacetic acid (2,4-D); N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (thidiazuron); N 6-(2-isopentenyl) adenine (2iP).

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Hector G. Nunez-Palenius, Daniel J. Cantliffe, Harry J. Klee, and Don J. Huber

Embryo abortion and empty seeds after self-pollination occur in some transgenic (ACO antisense) `Galia' male parental lines. An embryo-rescue system in this melon was developed to save potential viable embryos. To obtain the best and reliable embryo-rescue technique, several parameters were used including an improved (five new supplements) nutrient medium (named E-21) from the E-20A basic medium (Sauton and Dumax de Vaulx, 1987), an inoculation system (removing the embryo from the seed or intact seed), and the use of different fruit harvesting dates of the wild type and a transgenic `Galia' male parental line. Fruits of wild type (WT) and transgenic (ACO gene in antisense orientation) `Galia' male parental line were harvested at 4, 10, 17, 24, and 30 days after pollination (DAP). Fruits were surface sterilized by dipping in a 20% commercial bleach solution for 30 minutes. Subsequently, seeds were removed from fruit under sterile conditions. These seeds were either used to dissect the embryos or placed directly with the hilum facing E-20A or E-21 medium. Seedlings from all treatments were transferred to E-21 elongation medium, incubated 4 weeks, and transferred to soil to evaluate growth. The efficiency of this technique was greater when the time after pollination (4, 10, 17, 24, and 30 DAP) to rescue the embryos was increased. Thus, 30 DAP was the best time to rescue the embryos. The number of rescued embryos using E-21 medium was greater than with E-20A. We did not find any significant differences in survival efficiency rate between WT and transgenic embryos. We have obtained a competent embryo-rescue technique for WT and transgenic `Galia' male parental line, which can be applied to rescue valuable GMO hybrid-melon embryos.

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Yousef I. Dlaigan, A.E. Said, and M.A. El-Hamady

The effects of the physical state of nutrient media on the growth and elongation of excised date palm roots were investigated. Roots were cultured in a nutrient medium containing MS salts mixture, 1/2 modified White's organics, and (in mgliter–1): NaH2PO4 H2O, 170; sucrose, 60,000; inositol, 40; adenine sulfate, 240; activated charcoal, 4000; 2,4-D, 1; kinetin, 2. pH was adjusted at 5.7 ± 0.1. Both agar and Gelrite were singly used as solidifying agents. Liquid media were either stationary or rotated on gyratory shakers at 70 to 80 rpm. The effects of incubation of cultured roots under light or dark conditions were also studied. Media pH and its effects on growth and elongation of cultured roots were tested at various ranges (3, 4, 5, 6, 7, 8, 9, 10). Trials were also made to determine the passage length for transfer and subculture of cultured roots to a newly prepared medium. Liquid media highly supported better growth and elongation of cultured roots compared to solidified media. There was no significant difference in growth or elongation between agar or Gelrite media. Shaking liquid medium resulted in significantly better growth and elongation compared to stationary medium. No difference was observed between dark- and light-incubated cultured roots. Cultured roots grew and elongated better at pH 7.0–8.0. No growth or elongation occurred at pH 9.0. Roots continued to grow and elongate even after 12 weeks in culture. Therefore, 10 to 12 weeks after culture was determined to be the optimum passage length for date palm root culture.

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Rebecca E. Scoville and Todd P. West

The objective of this study was to investigate the effects of multiple nutrient salt formulations and different plant growth regulator concentrations on initiation and proliferation of axillary shoot culture of tropical hibiscus (Hibiscus rosa-sinensis L.). Combinations of five thidiazuron (TDZ) concentrations (0, 10-6, 10-7, 10-8, or 10-9 M) in conjunction with two 6-benzylaminopurine (BA) concentrations (0, 10-5 M) and two indole-3-butryic acid (IBA) concentrations (0, 10-5 M) were compared to determine which plant growth regulator combination(s) would stimulate the proliferation of the most viable axillary shoots. Also, five nutrient salt formulations (MS, 1/2 MS; Macro MS, WPM, LP, or DKW) ranging from high to low salt formulations were studied to determine a suitable nutrient medium formulation for axillary shoot proliferation. Nodal explants that were 2 cm in length were used to initiate cultures and were maintained on the various medium treatments plus 30 g·L-1 sucrose and 7 g·L-1 agar at a pH of 5.8. Explants were incubated about 30 cm beneath cool-white fluorescent lamps that provide a photon flux of about 40 μM·m-2·s-1 for a 16-hour photoperiod at 25 ± 3 °C. Nodal explants were transferred every 3 weeks for a total culture period of 12 weeks. At each transfer date data were collected on node number, axillary shoot number and length. Initial results indicate that high nutrient salt formulations coupled with low TDZ concentrations performed better at axillary shoot initiation. Poor shoot elongation was observed and further research needs to be performed to address this issue.

Open access

Jessica D. Lubell-Brand, Lauren E. Kurtz, and Mark H. Brand

Hyperhydricity of shoots initiated in vitro, poor shoot extension, inability of shoot cultures to maintain good growth over an extended time, and unsuccessful ex vitro rooting have limited the development of a commercial scale micropropagation system for hemp (Cannabis sativa). We present a culture initiation method that prevents shoot hyperhydricity using vented-lid vessels with 0.2-µm pores and medium containing agar at 1% (w/v). To optimize shoot multiplication in vitro, a control medium (medium A) and four treatment media (medium B, C, D, and E), with varying inorganic nutrients and vitamins were tested. Control medium A consisted of 1× Murashige and Skoog (MS) with vitamins plus 3% (w/v) sucrose, 0.5 mg·L−1 metatopolin, 0.1 mg·L−1 gibberellic acid, and 0.8% agar (w/v) at pH 5.7. The four treatment media differed from the control medium as follows: medium B, 2.5× MS with vitamins; medium C, 1× MS with vitamins plus added mesos [calcium chloride (anhydrous), magnesium sulfate (anhydrous), and potassium phosphate (monobasic) nutrients]; medium D, 1× MS with vitamins plus added vitamins; and medium E, 1× MS with vitamins plus added mesos and vitamins. Medium C and medium E produced more microcuttings than the control at 6 weeks after the initial subculture with shoot multiplication media and all other treatments at 9 and 12 weeks. Shoots grown on these two media displayed optimal extension and leaf lamina development; however, they exhibited slight chlorosis by 12 weeks after subculture with shoot multiplication media. In a separate experiment, medium E was supplemented with ammonium nitrate at 0, 500, 1000, or 1500 mg·L−1, and cultures grown with 500 mg·L−1 produced the most microcuttings and exhibited the best combination of shoot extension and leaf lamina development. We provide a method of prerooting microshoots in vitro that has resulted in 75% to 100% rooting ex vitro in rockwool. Using 10 recently micropropagated plants, ≈300 retip cuttings (cuttings taken from new shoots from recently micropropagated plants) were harvested over 10 weeks. The average weekly rooting was more than 90%. Retipping can produce nine-times as many plants in a similar amount of floor space as stem cuttings derived from traditional stock mother plants. The micropropagation/retipping method proposed can be a more efficient way to generate clonal liner plants for commercial-scale production.