Jeffrey Adelberg

Nutrient utilization in liquid/membrane system for watermelon micropropagation

Watermelon [Citrullus lanatus (Thunberg) Matsumura and Nakai] proliferating shoot meristems from established shoot cultures were inoculated on modified Murashige and Skoog salts medium supplemented with 10 μM 6-benzyladenine (BA) for shoot proliferation and on similar medium supplemented with 1 μM BA and 10 μM gibberellic acid (GA3) for shoot elongation. Agar-solidified medium and microporous polypropylene membrane rafts in liquid medium were used to support the tissues. Growth over culture time of proliferating and elongating tissues in liquid and agar-solidified media were compared. Nutrient depletion in liquid medium was monitored and quantified using ion selective electrodes. Tissue fresh weights in both proliferation and shoot elongation media were greater in liquid than in agar-solidified medium. Relative dry matter content, however, was greater in agar-solidified than in liquid medium. More shoots elongated in agar-solidified than in liquid medium. The numbers of buds or unelongated shoot meristems, however, were comparable for both the liquid and agar-solidified medium. Proliferating and elongating tissues in liquid medium used Ca++ and K+ minimally. NO3− was utilized but not depleted by proliferating tissues. NH4+, however, was depleted. Most of the NH4+ was utilized by the proliferating tissues within 21 days of culture when growth rate was greatest. At 35 days, residual Ca++, K+, NO3−, and NH4+ in proliferation medium were 81.0%, 67.8%, 55.7%, and 1.2% of initial levels, respectively. NO3− and NH4+ in shoot elongation medium were depleted. The greatest NO3− and NH4+ utilization was observed during the first 14 days of culture when the largest growth rate was obtained. The residual Ca++, K+, NO3− , and NH4+ in shoot elongation medium at 38 days were 63.5%, 37.9%, 21.2%, and 24.3% of initial concentrations, respectively. At the end of experiment, 72.3% and 42.8% of initial sugars were still remaining in the shoot proliferation and shoot elongation medium, respectively.

Spent medium analysis for liquid culture micropropagation of Hemerocallis on Murashige and Skoog medium

Residual nutrients from Murashige and Skoog medium were analyzed following a 5-wk multifactor experiment. Plant density, sugar concentration, and plant growth regulators (benzyladenine and ancymidol) were examined using four genotypes of daylily (Hemerocallis) to determine which factors most influenced nutrient use. Active nutrient uptake was observed for 11 nutrients (potassium, sodium, copper, phosphorus, iron, calcium, magnesium, manganese, boron, sulfur, and zinc) with lower concentrations in spent medium than in the tissue water volume (fresh-dry mass expressed as mL H2O). Two patterns of nutrient use were visualized by correlative analysis of nutrient uptake. Greatest growth lowered plant nutrient concentrations of potassium, sodium, phosphorus, iron, and copper in all genotypes, and luxuriant uptake was indicated with least growth. Potassium, sodium, iron, and copper concentrations in plant dry matter were equal to or exceeded what is observed in vigorously growing nursery plants. However, phosphorus concentration in plant dry matter was low enough to be considered deficient when compared to Hemerocallis plants in nursery production. With a second group of nutrients (calcium, magnesium, manganese, and boron), the genotype, “Barbara Mitchell” lacked active uptake and was deficient. Calcium concentration was low in all plants compared to Hemerocallis grown under nursery conditions (“Barbara Mitchell” was the lowest concentration) despite active uptake by the other three genotypes—“Brocaded Gown,” “Mary’s Gold,” and “Heart of a Missionary.” Magnesium concentration in these three genotypes was low enough in vessels with greatest growth to question its adequacy at high densities. Increased sucrose in medium reduced the dry matter concentrations of all tested nutrients. Plant growth regulators had less impact on nutrient use than genotype and plant density. Nutrient uptake may be an important physiological component of genotypic variation.

Sucrose Concentration in Liquid Media Affects Soluble Carbohydrates, Biomass and Storage Quality of Micropropagated Hosta

The effects of sucrose concentration (1, 3, 5, or 7% w/v) in liquid media, in the presence and absence of benzylaminopurine (BAP), on internal carbohydrate status and growth of Hosta tokudama Tratt. ‘Newberry Gold’ during the multiplication phase (stage II) was investigated. Cultures from all treatment combinations were transferred to media containing 3% (w/v) sucrose during the rooting phase (stage III). At the end of the stage III, these micropropagules were subjected to 5 weeks of storage at 10 °C under low light (photosynthetic photon flux of 5 µmol m−2s−1). Endogenous concentrations of soluble sugars (glucose, fructose, and sucrose) in the plantlets increased linearly as the media sucrose concentration increased from 1% to 7% during stage II. Root and shoot biomass increased with increasing media sucrose concentration. BAP increased the biomass and multiplication rate but did not affect internal concentration of soluble sugars. While in storage, endogenous sugar levels and plantlet dry weight remained unchanged for all treatments. Following storage, plants originally cultured in 5% and 7% media sucrose had higher dry weight and less leaf chlorosis than those cultured in 1% and 3% media. Differences in endogenous soluble sugar levels at the end of stage III rooting, and after storage were related to the sucrose concentration of the initial stage II multiplication medium. Increased media sucrose levels during the multiplication cycle has a positive, long-term effect on plant morphology and quality.

Efficiency in thin-film liquid system for Hosta micropropagation

AbstractThree varieties of Hosta (‘Striptease’, ‘Minuteman’ and ‘Stiletto’) at four densities (40, 80, 120 and 200 explants per litre) were micropropagated on semi-solid agar and a thin-film liquid system with intermittent wetting of plant tissue. The mechanics of wetting by a small wave front required a larger rectangular vessel (11 × 27=297 cm 2) compared to the common cylindrical baby food jar (18 cm 2). Plants multiplied more rapidly in the agitated thin-film system than on agar. Lower plant densities increased rates of multiplication in liquid, but had little or no effect on multiplication rate on agar. Increasing plant density lowered the overall multiplication rate, but yielded greater numbers of plants per vessel. Yield, tabulated for utilization of shelf-space in growth room, was greater at all densities in rectangular vessels of liquid than conventional jars of agar media. Increased plant density lowered the sugar residual in media following the culture cycle and liquid media had less residual sugar than agar media. A liquid medium with 50 g l−1 sucrose was concentrated enough so that sugar depletion did not limit growth, even at the highest densities. The liquid system allows the technician to skip the step of manually spacing and orienting the freshly cut bud tissue at the transfer station. Harvesting 75–100 plants per vessel from the large rectangular vessels resulted in most efficient use of technician time. Plants from liquid and agar acclimatized to greenhouse. Increased multiplication, space utilization, sugar availability and worker efficiency was demonstrated to be greater in thin-film liquid than more conventional agar-based system.

SIVB 2003 Congress Symposium Proceeding: Plant Growth and Sugar Utilization in an Agitated, Thin-Film Liquid System for Micropropagation

SummaryAgitated layers of liquid medium were created on platform shakers in jars with 25–30 ml of medium (similar to conventional agar culture) rotating at 90 rpm. Thin films were scaled up in larger rectangular vessels on tilted shelves that periodically rock. In jars of liquid medium with a density of 180 explants per liter, multiplication rates of Hota tokudama var. ‘Newberry Gold’ were optimal with a media sucrose concentration of 5% [both with and without 1 μM benzyladenine (BA)]. Endogenous levels of soluble sugars were directly related to the concentration of sucrose in the medium. Three Hosta cultivars (‘Striptease’, ‘Minuteman’, and ‘Stiletto’) with plant densities of 40–200 explants per liter of medium were tested in larger, agitated, thin-film vessels in media with 5% sucrose and directly compared to agar medium. Higher rates of multiplication were observed in liquid than agar with the magnitude of the difference dependent on explant density. Pooled results for the three varieties with 200 explants per liter showed multiplication rates of 1.7x and 2.3x for agar and thin-film liquid, respectively. At 40 explants per liter, the multiplication rate was increased to 2.1x for agar and 3.4x for thin-film liquid. Sugar uptake was greater in liquid than agar and was greater in the higher densities, with the magnitude of the effect dependent on plant variety. Increased vessel size in the liquid, thin-film system and greater sugar uptake allowed more, larger plants to be harvested. Alocasia macrorrhizos was cultured in growth medium containing 1μM BA and 5% sucrose with plant densities in the range of 33–330 explants per liter. Dry weight and multiplication rate were greater in the liquid system than agar with the magnitude of the difference dependent on plant density. With approximately 165 explants per liter, and greater at the initiation of culture, plant density limited growth in both agar and liquid thin-film systems. In a multiplication medium (3 μM BA and 3 μM ancymidol) plant size was reduced by 50% and 60% (fresh weight) in liquid and agar, respectively. Initial density in the range of 165–330 explants per liter did not limit growth with the smaller plants in liquid or semisolid multiplication medium. Sugar uptake was greater in liquid than agar. While ample sugar was present in media for growth at any density on agar, sugar depletion was limiting growth at highest densities with the larger plants in liquid growth medium. In semisolid agar medium, sugar uptake by plants was more rapid than diffusion across the agar medium, resulting in non-equilibrium conditions following the culture cycle. In agitated, liquid medium, a greater transfer of sugars to plant tissue was related to accelerated growth.

Photoautotrophic shoot and root development for triploid melon

The aim of this investigation was to establish environmental factors which promote growth and photosynthesis of melon (Cucumis melo L.) shoot buds, in vitro, and determine if photoautotrophic shoots had superior root forming ability in photoautotrophic environments. Buds from the triploid melon clone ‘(L-14×B)×L-14’ were observed for 21 days after transfer from a multiplication MS medium with 3% sucrose and 10 μM benzyladenine (BA) to a shoot development medium with 1 μM BA at three levels of sucrose in the medium (0, 1 and 3%), and light (50, 100 and 150 PPF) and CO2 (500, 1000 and 1500 ppm) in the culture chamber. More shoot buds were observed with 3% sucrose in the medium. Increased light and CO2 had a positive interaction with shoot proliferation. Fresh and dry weights were greatest at 3% sucrose, 150 PPF light and 1500 ppm CO2. Shoot buds grew more slowly in sugar-free medium, but fresh and dry weight still doubled over 21 days of culture. Net photosynthetic rates (NPR) of buds were negative after four days in treatment conditions, but became positive after transfer to fresh, sugar-free medium. Two triploid genotypes of melon were (1) grown in vitro with sugar (photomixotrophic) and without sugar (photoautotrophic), (2) rooted in sugar-free media, both in a laboratory controlled environment chamber (in vitro) and a greenhouse acclimatization unit (ex vitro), and (3) compared for subsequent nursery growth in the greenhouse unit. The genotype ‘(L-14×B)×L-14’ produced more shoots than ‘(L-14×B)×Mainstream’ in both photomixotrophic or photoautotrophic conditions. ‘(L-14×B)×L-14’ rooted as well from either photoautotrophic and photomixotrophic shoots but ‘(L-14×B)×Mainstream’ rooted less frequently from photoautotrophic shoots. Seventy-six percent of the shoots in the laboratory controlled environment chamber were able to root photoautotrophically, whereas 47% of the shoots in the greenhouse acclimatization unit were rooted. Between 77% and 88% of plantlets from all treatment combinations survived transfer to the nursery. After growth in the nursery, the sizes of plants (fresh weight, dry weight, leaf area) were the same for either genotype, from either photoautotrophic or photomixotrophic shoots. Nursery plants that had been rooted in the laboratory controlled environment chamber were larger than those rooted in the acclimatization greenhouse chamber.

AGITATED, THIN-FILMS OF LIQUID MEDIA FOR EFFICIENT MICROPROPAGATION

In vitro culture is a semi-closed system that aseptically provides oxygen, water, organic carbon source (and/or CO2 and light), nutrients, and plant growth regulators (PGR), at a controlled temperature. A traditional view of plant tissue culture involves placing a small piece of tissue on the gelled-media surface, in a jar, plate or tube, and allows exponential growth unfettered by lack of resource in a uniform microenvironment. Many reports summarized in this volume show increased productivity (per plant, unit area or time) were achieved with larger vessels of liquid medium yielding greater numbers and / or larger plants. Liquid systems that improve distribution of dissolved nutrients, water and oxygen, in the vessel stimulate growth of plant tissues. Simplicity, cost and ergonomic factors are human constraints imposed on designs intended for commercial use. This chapter describes a hybrid micropropagation process that invokes features of semi-solid gel and bioreactor technology. The agitated, thin-film system (or rocker) uses large, rigid rectangular vessels in a slow pitching motion to intermittently wet and aerate plantlets [1] (see Figure 1). Economy of scale was optimized for the twodimensional growth surface area in the vessel. Gentle oxygenation of liquid media was similar to wave machines Eibl and Eibl describe for cell and tissue culture in Part 2 of this volume. Shoot surfaces, intermittently wet or dry in a large headspace, accumulate large quantities of solutes from media resulting in high shoot quality similar to temporary immersion systems. Vessel and culture room designs differ from conventional micropropagation, or the bioreactors discussed in other chapters of Part 2. The first section of this chapter discusses nutrients and heterotrophic growth in agar and liquid;the secondsectioncomparesefficiencyof agitated,thin-film processwith agar-based media system; and third one lists designconsiderations for the vessel and culture shelves in the growth room during scale-up. Comparisons will be drawn to agar-gelled media in small round jars, typical of many micropropagation protocols using semi-solid media.

Sucrose in storage media and cultivar affects post-storage regrowth of in vitro Hosta propagules

The goal of this research was to investigate if culturing in high sucrose (5%) liquid media during multiplication phase (stage II) would enhance endogenous sugar levels and dry matter sufficiently to allow storage of in vitro plants in sugar free media without adversely affecting post-storage recovery. Hosta tokudama ‘Newberry Gold (NBG)’ and Hosta ‘Striptease’ were cultured in Murashige and Skoog (MS) media containing 5% sucrose during stage II and transferred to rooting phase (stage III) in MS medium without (0%) sucrose or with 3% sucrose for 4 weeks. At the end of stage III, cultures were stored, with the remaining media, at 10 °C with 5 μmol m−2 s−1 photosynthetic photon flux (PPF) from cool white fluorescent lamps for 7 or 14 weeks with or without a 2-week dark period prior to removal from storage. In both cultivars, stage III plants cultured in 3% sucrose media had higher soluble sugar levels and greater shoot and root biomass than those cultured in 0% sucrose media. Shoot and root soluble sugars decreased during storage. Shoot growth ceased during storage in both media. Root dry matter continued to increase in plants stored in 3% sucrose media but did not change in 0% sucrose media. Plants cultured in 3% sucrose media had less leaf chlorosis and less mortality after 7 or 14 weeks of low temperature storage than the plantlets from sugar free media. Extending the storage period from 7 to 14 weeks or introduction of 2-week dark period at the end of storage did not affect leaf chlorosis or plant mortality during acclimatization. Post-storage growth varied with the cultivar. Benefit of having sucrose in storage media was to develop a strong root system that aided the acclimatization and post-storage growth following 7 or 14 week storage. Sucrose ‘loading’ by culturing plants in liquid media containing 5% sucrose did not allow storage in sugar free media without adversely affecting post-storage growth in both cultivars.

In vitro growth of Curcuma longa L. in response to five mineral elements and plant density in fed-batch culture systems.

Plant density was varied with P, Ca, Mg, and KNO3 in a multifactor experiment to improve Curcuma longa L. micropropagation, biomass and microrhizome development in fed-batch liquid culture. The experiment had two paired D-optimal designs, testing sucrose fed-batch and nutrient sucrose fed-batch techniques. When sucrose became depleted, volume was restored to 5% m/v sucrose in 200 ml of modified liquid MS medium by adding sucrose solutions. Similarly, nutrient sucrose fed-batch was restored to set points with double concentration of treatments’ macronutrient and MS micronutrient solutions, along with sucrose solutions. Changes in the amounts of water and sucrose supplementations were driven by the interaction of P and KNO3 concentrations. Increasing P from 1.25 to 6.25 mM increased both multiplication and biomass. The multiplication ratio was greatest in the nutrient sucrose fed-batch technique with the highest level of P, 6 buds/vessel, and the lowest level of Ca and KNO3. The highest density (18 buds/vessel) produced the highest fresh biomass at the highest concentrations of KNO3 and P with nutrient sucrose fed-batch, and moderate Ca and Mg concentrations. However, maximal rhizome dry biomass required highest P, sucrose fed-batch, and a moderate plant density. Different media formulations and fed-batch techniques were identified to maximize the propagation and storage organ responses. A single experimental design was used to optimize these dual purposes.

Effects of Cytokinin on Multiplication and Rooting of Aloe barbadensis during Micropropagation on Agar and Liquid Medium

Aloe barbadensis (syn. Aloe vera) was micropropagated on agar and liquid medium at varied benzyladenine (BA) and meta-topolin (MT) concentrations (0, 1, 3.2, and 10 μM) for three successive culture cycles and then transferred to a greenhouse for growth. MT induced multiplication at the highest concentration (10 μM) and BA produced the greatest number of plantlets (at 3.2 μM) with optimal multiplication at approximately 6 μM. Liquid medium did not affect multiplication rate when compared with agar, but plants were twice as large from liquid as compared with those from agar at the time of transfer to the greenhouse. After five weeks of growth, plants in the greenhouse micropropagated on liquid culture were still larger than plants micropropagated on agar with BA and MT. A carryover of cytokinin inhibited rooting, and plants on agar were more severely affected than plants on the liquid medium. Cytokinin carryover reduced rooting from 92% (control) to 68% with either the 3.2 μM MT or 10 μM BA and at 10 μM MT only about 20% of the aloe plants rooted. There appeared to be a trade-off between maximum multiplication rates and best plant quality for ex vitro transfer. Using liquid medium led to larger plants and lessened the cytokinin carryover effect on rooting without affecting the multiplication rate. Approximately 6 μM BA in liquid medium would be optimal for multiplication and rooting of A. barbadensis. Introduction Aloe barbadensis Mill. (syn. Aloe vera L.) is a xerophyte native to Africa and Arabia, but the exact origin is disputed. The aloe plant will not withstand a hard freeze and only grows in a climate similar to that of the Mediterranean area with mild wet winters and hot dry summers. While the plant can tolerate drought, no juice will be produced if the weather is too dry. Aloe flourishes in well-drained fertile soil and requires about 1 m 2 of field space for growth and development. While the demand for aloe is increasing, cultivation is slow as seed does not often form on plants in cultivation, and when available, these seeds can take years to germinate. Propagation is typically done from offsets, although these take two to three years of field growth to reach a harvestable size. Vegetative propagation is generally too slow for cultivation purposes. Successful micropropagation of aloe would minimize the large requirements for time and space in field propagation and has been proposed to meet the need for large numbers of plants (Ken Altman, personal communication). In commercial horticulture, micropropagation usually involves enhanced axillary divisions and is most often induced by the synthetic cytokinin, benzyladenine (BA). BA retained in plants during subsequent transfers, known as cytokinin-carryover, lowers the rooting response in the greenhouse. For example, BA accumulated in the basal portion of Adelberg and Naylor-Adelberg: Effects of Cytokinin on Multiplication and Rooting of Aloe barbad