Young Reproductive Structures Promote Nitrogen Fixation in Soya Bean

In many legumes the transition from the vegetative to the reproductivephase of development is associated with a marked increase inthe rate of symbiotic nitrogen fixation. In soya bean [Glycinemax (L.) Merr.), the removal of reproductive parts at differentstages of their development showed that the increase in nitrogenfixation rate was primarily due to the presence of flower buds.The increase in the fixation rate of intact reproductive plantswas accompanied by a rapid increase in the weight of noduleson lateral roots and it is suggested that these nodules areresponsible for much of the nitrogen fixation which occurs duringreproductive growth. Maintaining plants in the vegetative stateprovided evidence which suggests that it is the flower budsand not the flowering stimulus which are responsible for theincrease in fixation rate. The marked effects on vegetativegrowth of removing reproductive parts suggests that the mechanisminvolved in the promotion of nitrogen fixation may be hormonal. Glycine max (L.) Merr., soya bean, nitrogen fixation     

In vitro Culture of Immature Cotyledons of Soya Bean (Glycine max L. Merr.)

An in vitro procedure promoting the rapid growth and proteinincrease of soya bean cotyledons has been developed. The amountof protein synthesized varied greatly depending on the nitrogen(N) source provided. Glutamine was the most effective N source,while inorganic forms of N were ineffective. Growth and proteinsynthesis were both more rapid in vitro than in vivo. Underthe best conditions, soya bean cotyledons increased 8-fold bothin dry weight and in protein in 6 days. The formation of the7S and 11S storage proteins in vitro was similar to that invivo. Hence, this in vitro culture method is appropriate forstudying legume seed storage protein synthesis under controlledconditions.     

Studies on Genetic Male-Sterile Soybeans : IV. Effect of Male Sterility and Source of Nitrogen Nutrition on Accumulation, Partitioning, and Transport of Nitrogen

Soybean (Glycine max [L.] Merr.) germplasm, isogenic except for loci controlling male sterility (ms₁) and nodulation (rj₁), was used to investigate the effects of reproductive tissue development and source of nitrogen nutrition on accumulation, transport, and partitioning of nitrogen in a greenhouse experiment. Nodulated plants were supplied nitrogen-free nutrient solution, and nonnodulated plants were supplied nutrient solution containing 20 millimolar KNO₃. Plants were sampled from flowering until maturity (77 to 147 days after transplanting).

Accumulation rates of nitrogen in whole plants during reproductive growth were not significantly different among the four plant types. Nitrogen accumulation in the sterile, nonnodulated plants, however, ceased 2 weeks earlier than in fertile, nonnodulated or fertile and sterile, nodulated plants. This early cessation in nitrogen accumulation resulted in sterile, nonnodulated plants accumulating significantly less whole plant nitrogen by 133 days after transplanting (DAT) than fertile, nonnodulated plants. Thus, changing the site of nitrogen assimilation from nodules (N₂-fixing plants) to roots and leaves (NO₃-fed plants) resulted in similar whole-plant nitrogen accumulation rates in fertile and sterile plants, despite the absence of seed in the latter.

Leaflet and stem plus petiole tissues of both types of sterile plants had significantly higher nitrogen concentrations after 119 DAT than both types of fertile plants. Significantly higher concentrations and exudation rates of nonureide, reduced-nitrogen in xylem sap of sterile than of fertile plants after 105 DAT were observed. These latter results indicated possible cycling of nonureide, reduced-nitrogen from the downward phloem translocation stream to the upward xylem translocation stream in roots of sterile plants. Collectively, these results suggest a lack of sinks for nitrogen utilization in the shoots of sterile plants. Hence, comparison of nitrogen accumulation rates for sterile and fertile plants does not provide a definitive test of the hypothesis that reproductive tissue development limits photosynthate availability for support of N₂ fixation and nitrate assimilation in determinate soybeans.

Nitrogen assimilation during reproductive growth met a larger proportion of the reproductive-tissue nitrogen requirement of nitrate-dependent plants (73%) than of N₂-fixing plants (63%). Hence, vegetative-tissue nitrogen mobilization to reproductive tissue was a more prominent process in N₂-fixing than in nitrate-dependent plants. N₂-fixing plants partitioned nitrogen to reproductive tissue more efficiently than nitrate-dependent plants as the reproductive tissues of the former and latter contained 65 and 55%, respectively, of the whole-plant nitrogen at the time that nitrogen accumulation in reproductive parts had ceased (133 DAT).

     

Dynamics of Carbon Photosynthetically Assimilated in Nodulated Soya Bean Plants under Steady-state Conditions 1. Development and Application of 13CO2 Assimilation System at a Constant 13C Abundance

A long-term, steady-state ¹³CO₂ assimilation system at a constantCO₂ concentration with a constant ¹³C abundance was designedand applied to quantitative investigations on the allocationof photoassimilated carbon in nodulated soya bean (Glycine maxL.) plants. The CO₂ concentration in the assimilation chamberand its ¹³C abundance were maintained constant with relativevariances of less than ±0.5 per cent during an 8-h assimilationperiod. At the termination of 8-h ¹³CO₂ assimilation by plantsat early flowering stage, the currently assimilated carbon relativeto total tissue carbon (measured by the degree of isotopic saturation)were for young leaves (including flower buds), 13.9 per cent;mature leaves, 15.7 per cent; stems+petioles, 5.9 per cent;roots, 5.4 per cent and nodules, 6.9 per cent, 48 h after theend of the ¹³CO₂ assimilation period, they were 12.3, 7.5, 7.4,6.8 and 6.1 per cent, respectively. The treatment with a highconcentration of nitrate in the nutrient media significantlydecreased the allocation of ¹³C into nodules. Experiments on¹³CO₂ assimilation by plants at the pod-filling stage were alsoconducted. Labelling by ¹³C was weaker than at the early floweringstage, but an intense accumulation of ¹³C into reproductiveorgans was observed. Glycine max L., nodulated soya bean plants, ¹³CO₂ assimilation, carbon dynamics     

Comparisons of the Respiratory Effluxes of Nodules and Roots in Six Temperate Legumes

The specific respiration rates of nodulated root systems, ofnodules and of roots were determined during active nitrogenfixation in soya bean, navy bean, pea, lucerne, red clover andwhite clover, by measurements on whole plants before and afterthe removal of nodule populations. Similar measurements weremade on comparable populations of the six legumes, lacking nodulesbut receiving abundant nitrate-nitrogen, to determine the specificrespiration of their roots. All plants were grown in a controlled-environmentclimate which fostered rapid growth. The specific respiration rates of nodulated root systems ofthe three grain and three forage legumes during a 7–14-dayperiod of vegetative growth varied between 10 and 17 mg CO₂g^–1 (dry weight) h^–1. This mean value consistedof two components: a specific root respiration rate of 6–9mg CO₂ g^–1 h^–1 and a specific nodule respirationrate of 22–46 mg CO₂ g^–1 h^–1. Nodule respirationaccounted for 42–70 per cent of nodulated root respiration;nodule weight accounted for 12–40 per cent of nodulatedroot weight. The specific respiration rates of roots lackingnodules and utilizing nitrate nitrogen were generally 20–30per cent greater than the equivalent rates of roots from nodulatedplants. The measured respiratory effluxes are discussed in thecontext of nitrogen nitrogen fixation, nitrate assimilation. Glycine max, Phaseolus vulgaris, Pisum sativum, Medicago sativa, Trifolium pratense, Trifolium repens, soya bean, navy bean, pea, lucerne, red clover, white clover, nodule respiration, root respiration, fixation, nitrate assimilation     

Effect of Source of Nitrogen on the Growth of Fiskeby Soya Bean: the Carbon Economy of Whole Plants

Fiskeby V soya bean was grown from seed germination to seedmaturation with two contrasting patterns of nitrogen metabolism:either wholly dependent on dinitrogen fixation, or with an abundantsupply of nitrate nitrogen, but lacking root nodules. The carbonand nitrogen economies of the plants were assessed at frequentintervals by measurements of photosynthesis, shoot and rootrespiration, and organic and inorganic nitrogen contents. Plantsfixing atmospheric nitrogen assimilated only 25–30 percent as much nitrogen as equivalent plants given nitrate nitrogen:c. 40 per cent of the nitrogen of ‘nitrate’ plantswas assimilated after dinitrogen fixation had ceased in ‘nodulated’plants. The rates of photosynthesis and respiration of the shootsof soya bean were not markedly affected by source of nitrogen;in contrast, the roots of ‘nodulated’ plants respiredtwice as rapidly during intense dinitrogen fixation as thoseof ‘nitrate’ plants. The magnitude of this respiratoryburden was calculated to increase the daily whole-plant respiratory loss of assimilate by 10–15 per cent over thatof plants receiving abundant nitrate. It is concluded that ‘nodulated’plants grew more slowly than ‘nitrate’ plants inthese experiments for at least two reasons: firstly, the symbioticassociation fixed insufficient nitrogen for optimum growth and,secondly, the assimila tion of the nitrogen which was fixedin the root nodules was more energy-demanding in terms of assimilatethan that of plants which assimilated nitrogen by reducing nitratein their leaves.     

Effects of Temperature and Photoperiod on Flowering in Soya bean [Glycine max (L.) Merrill]: a Quantitative Model

Factorial combinations of five photoperiods (8 h 20 min, 10h, 11 h 40 min, 13 h 20 min and 15 h) and three night temperatures(14, 19 and 24 C) combined with a single day temperature (30C) were imposed on nodulated plants of nine soya bean genotypes[Glycine max (L.) Merrill] grown in pots in growth cabinets.The times to first appearance of open flowers were recorded.For a photoperiod-insensitive cultivar, and for the remainingeight photoperiod-sensitive genotypes in photoperiods shorterthan the critical daylength, the rates of progress towards flowering(the reciprocals of the times taken to flower) were linear functionsof mean diurnal temperature. For all photoperiod-sensitive genotypes,times to flowering in photoperiods longer than the criticaldaylength increased as inverse functions of both increasingphotoperiod and decreasing temperature. A consequence of thesetwo relations is that the critical daylength becomes longerwith higher mean temperatures. In the five photoperiod-sensitivegenotypes which flowered in all environments before the experimentwas terminated (after 150 d) the delays in flowering due tolow temperatures or long photoperiods were limited by a maximumperiod to flowering specific for each genotype. These resultsare discussed in relation to the development of a simple techniquefor the large-scale screening of soya bean germplasm to determinephoto-thermal response surfaces for flowering. Glycine max (L.) Merrill, soya bean, flowering, photoperiod, temperature, screening, germplasm     

Turnover of Storage Protein in Seeds of Soya Bean and Pea

We were interested in determining whether the low protein contentof pea seeds (Pisum sativum L.) as compared to soya bean seeds(Glycine max L. Merrill) might be due to faster degradationof the pea storage proteins during development of the seed.Pea and soya bean cotyledons were subjected to a ‘pulse-chase’experiment using [³H]glycine in in-vitro cultures. In peas,legumin had a half-life of 146 days, while vicilin had a half-lifeof 39 days. There was no measureable degradation of soya beanstorage proteins. Even with the pea storage proteins, the half-liveswere so much longer than the maturation time of seeds that degradationof storage proteins could not account for the lower proteincontent of peas as compared to soya beans. The validity of theseresults was indicated by the finding that non-storage proteinshad much shorter half-lives and that omission of a carbon ora nitrogen source greatly accelerated degradation. Labelledglycine was found to be a good probe for protein turnover studiesbecause it was very rapidly metabolized. Glycine max L. Merrill, soya bean, Pisum sativum, L. pea, protein turnover, storage proteins, legumin, vicilin     

Effects of Nitrate Level on Nitrogen Metabolism in Winged Bean and Soya Bean

The effects of high (15 mM) and low (0.75 mM) solution nitratelevels on nitrogen metabolism in three genotypes (IL 7A, IL13 and IL 21) of winged beans [Psophocarpus tetragonolobus (L.)DC.] and one genotype (Williams) of soya bean [Glycine max (L.)Merrill] were investigated. Plants were grown for 42 days ina greenhouse in solution culture prior to sampling. The 15 mM nitrate treatment resulted in greater growth of allplant parts except roots. Growth of soya beans was more responsiveto nitrate level than was growth of winged beans. The high nitratelevel inhibited nodulation in all plants. The IL 13 and IL 21winged bean genotypes had similar nitrogenase activity (acetylenereduction per plant) as the soya bean and IL 7A winged beangenotype had lower activity. However, the IL 13 winged beangenotype had higher nitrogenase activity (acetylene reductionper unit nodule mass) than the other three genotypes which allhad similar activity. The 15 mM solution nitrate level stimulatedleaf and root nitrate reductase (NR) activity for all plants.All winged bean genotypes had higher leaf NR activity and higherpercentage reduced- and nitrate-nitrogen contents of leavesand stems compared with soya beans. However, total protein (reducednitrogen) was greater in soya beans when sampled indicatingthat more nitrate had been metabolized by soya beans than bywinged beans during the 42-day growth period. Psophocarpus tetragonolobus (L.) DC., winged bean, Glycine max (L.) Merrill, Soya bean, nitrate reductase, nitrogen fixation, nitrogenase activity, nodulation     

Control of Seed Growth in Soya Beans [Glycine max (L.) Merrill]

The seed is the primary sink for photosynthate during reproductivegrowth and an understanding of the mechanisms controlling therate of seed growth is necessary to understand completely theyield production process. The growth rate of individual seedsof seven soya bean [Glycine max (L.) Merrill] cultivars withgenetic differences in seed size varied from 10.8 to 3.9 mgseed^–1 day^–1. The growth rates were highly correlatedwith final seed size. The growth rate of cotyledons culturedin a complete nutrient medium was highly correlated with thegrowth rate of seeds developing on the plant and with finalseed size. The number of cells per seed in the cotyledons variedfrom 10.2 to 5.7 x 10⁶ across the seven cultivars. The numberof cells per seed in the cotyledons was significantly correlatedwith final seed size and the seed growth rate both on the plantand in the culture medium. The data suggest that genetic differencesin seed growth rates are controlled by the cotyledons and thenumber of cells in the cotyledons may be the mechanism of control. Glycine max L., soya bean, seed size, growth rate, cell number, sink activity     

Acclimation of Photosynthate Partitioning and Photosynthetic Rates to Changes in Length of the Daily Photosynthetic Period

Pangola, soya bean and spinach plants were grown in long andshort day photosynthetic periods. Reciprocal shifts betweenlong and short day grown plants were made to study acclimationin the rate of leaf starch synthesis with change in daylength.The rate of leaf starch accumulation is a function of the lengthof the daily photosynthetic period. Acclimation, that is a changein partitioning with a change in length of the photosyntheticperiod, occurs in a variety of species. Acclimation in the rateof starch accumulation occurs rapidly in pangola and is apparentlycomplete the day after a change in length of the daily photosyntheticperiod. Soya bean and spinach leaves require a few days in thenew environment for an acclimation to occur. Digitaria decumbens Stent., Glycine max (L.) Merr., Spinacia oleracea L., pangola, soya bean, spinach, specific leaf weight, starch, photosynthesis     

Assimilation and Translocation in Perennial Grasses

Single leaves, ears, or shoots of timothy (Phleum pratense L.)were exposed in light to ¹⁴CO₂, then left overnight, after whichthe plants were autoradiographed. The following conclusionswere drawn. Actively growing leaves retain all their assimilatesand import from older ones. Fully expanded leaves export butdo not import assimilates. Export begins before leaf expansionis complete, so import and export may for a time be simultaneous.Exports go at first to younger leaves and to roots, accumulatingat meristems. At later stages, exports move downwards ratherthan upwards. Buds and small tillers import from older shoots,but large tillers do not import from other shoots or exportto other large ones. Ears assimilate while still green, andimport assimilates from their associated flag leaves. Exportsfrom other leaves on flowering stems move downwards. These findings agree in general with those from other plants:they are discussed in relation to the vascular system of thegrass plant, and the need for further studies, particularlyquantitative ones, is emphasized.     

Mineral Depletion and Leaf Senescence in Soya Bean as Influenced by Foliar Nutrient Application During Seed Filling

Senescence, as judged by the time courses of leaf lamina photosynthesis,soluble protein and chlorophyll contents, was studied in relationto mineral redistribution in field-grown soya beans [Glycinemax (L.) Merr] to investigate the hypothesis that the depletionof nutrients m the leaves by the developing seeds is the causeof soya bean senescence. A mineral nutrient solution was appliedto the canopy during the seed-filling period, and the effectson senescence and mineral depletion of the leaves were determinedin three cultivars, at two leaf positions, weekly from beginningof seed filling through physiological maturity. The onset of senescence occurred shortly after the beginningof rapid seed filling Photosynthetic rate declined about 60per cent within 3 weeks. Protein dropped by 52 per cent andchlorophyll by 48 per cent over the same period. Foliar nutrient application, at a rate previously shown to givesignificant yield increases in soya beans, increased the concentrationsof N, P and K in the leaf laminae, but tended only to delaytheir decline and failed to either delay the onset or alterthe course of senescence. The results of this experiment seem to indicate that, undernormal growth conditions, the events of senescence in the soyabean are not causally related to the N, P or K concentrationsof the leaf laminae Glycme max (L.) Merr., soya bean, nitrogen, phosphorus, potassium, leaf protein, chlorophyll, photosynthesis, foliar nutrient application, mineral depletion, leaf senescence     

CRISPR/Cas9‐mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean

Flowering is an indication of the transition from vegetative growth to reproductive growth and has considerable effects on the life cycle of soya bean (Glycine max). In this study, we employed the CRISPR/Cas9 system to specifically induce targeted mutagenesis of GmFT2a, an integrator in the photoperiod flowering pathway in soya bean. The soya bean cultivar Jack was transformed with three sgRNA/Cas9 vectors targeting different sites of endogenous GmFT2a via Agrobacterium tumefaciens‐mediated transformation. Site‐directed mutations were observed at all targeted sites by DNA sequencing analysis. T1‐generation soya bean plants homozygous for null alleles of GmFT2a frameshift mutated by a 1‐bp insertion or short deletion exhibited late flowering under natural conditions (summer) in Beijing, China (N39°58′, E116°20′). We also found that the targeted mutagenesis was stably heritable in the following T2 generation, and the homozygous GmFT2a mutants exhibited late flowering under both long‐day and short‐day conditions. We identified some ‘transgene‐clean’ soya bean plants that were homozygous for null alleles of endogenous GmFT2a and without any transgenic element from the T1 and T2 generations. These ‘transgene‐clean’ mutants of GmFT2a may provide materials for more in‐depth research of GmFT2a functions and the molecular mechanism of photoperiod responses in soya bean. They will also contribute to soya bean breeding and regional introduction.     

Carbon Metabolism, Nitrogen Assimilation, and Seed Yield of Cowpea (Vigna unquiculata L. Walp.) Grown in an Adverse Temperature Regime

When grown in an environment known not to favour the productionof large seed yields (warm days-cool nights; 33–19 °C),non-nodulated plants of cowpea cv. K 2809 supplied with abundantinorganic nitrogen not only assimilated N more rapidly but alsoproduced larger total dry weights and seed yields than plantsdependent on Rhizobium CB 756. Remobilization of nitrogen fromvegetative organs started sooner in nitrate-dependent than innodulated plants and contributed 69 and 47%, respectively, tothe N content of mature fruits. Plants dependent on nodulesrelied more on current assimilation of nitrogen during the laterstages of fruit growth than those given inorganic N; they alsoutilized a larger proportion of shoot-derived photosynthatesin growth of organs below ground and in the respiratory activitiesof both nodules and supporting roots. Although nitrate-dependentplants developed larger shoot systems than those relying onnodules, the distribution of carbon and nitrogen to leaves decreasedmarkedly as branches extended during early reproductive growth.The respiration of roots on nodulated plants became more efficientduring the later stages of fruit growth whereas the populationof secondary nodules present at this stage of development respiredless efficiently (mg C consumed per mg N assimilated) than theprimary nodules present earlier during development.     

Dynamics of Carbon Photosynthetically Assimilated in Nodulated Soya Bean Plants under Steady-state Conditions 2. The Incorporation of 13C into Carbohydrates, Organic Acids, Amino Acids and some Storage Compounds

Nodulated soya bean (Glycine max L.) plants at the early floweringstage were allowed to assimilate ¹³CO₂ under steady-state conditions,with a constant ¹³C abundance, for 8 h in the light. The plantswere either harvested immediately or 2 d after the end of the¹³CO₂ feeding, divided into young leaves (including flower buds),mature leaves, stems+petioles, roots and nodules; the ¹³C abundancein soluble carbohydrates, organic acids, amino acids, starchand poly-ß-hydroxybutyric acid was determined witha gas chromatography-mass spectrometry. The rapid turnover of ¹³C in the sucrose pools observed in allorgans of the plants showed that sucrose was the principal materialin the translocation stream of primary products of photosynthesis.At the end of the ¹³CO₂ exposure, sucrose in the mature leavesas the major source organs and in the stems+petioles was labelledwith currently assimilated carbon to about 75 per cent, whereasa much higher labelling of sucrose was found in the roots andin the nodules. This suggests the existence of two or more compartmentedpools of sucrose in mature leaves and also in stems+petioles. The relative labelling patterns of individual organic acidsand amino acids were similar in various plant organs. However,the rapid turnover of succinate and glycine was characteristicof nodules. Treatment with a high concentration of nitrate inthe nutrient media increased the turnover rate of amino acidcarbon in shoot organs and roots, while it markedly decreasedthe labelling of amino acids in nodules. The cyclitols, exceptfor D-pinitol, were significantly labelled with assimilated¹³C in mature leaves, but in nodules, the labelling was verymuch less. In the nodules, which were actively fixing atmospheric nitrogen,a large proportion (80–90 per cent) of currently assimilatedcarbon was found as sucrose and starch at the end of the ¹³CO₂feeding. This was also true of the roots. On the other hand,in young growing leaves, the distribution of currently assimilatedcarbon into sucrose, starch and other soluble compounds wasmuch less. This suggests that a large amount of carbon assimilatedby and translocated to young leaves was used to make up structuralmaterials, mainly protein and cell wall polymers synthesis,during the light period. Glycine max L., soya bean, ¹³CO₂ assimilation, carbon metabolism in nodules     

Assimilation of Ammonium Nitrogen by Heterotrophic and Symbiotrophic White Lupine Plants

The activities of glutamate dehydrogenase, asparagine synthetase, and total glutamine synthetase in the organs of the white lupine (Lupinus albus L.) plants were measured during plant growth and development. In addition, the dynamics of free amino acids and amides in plant organs was followed. It was shown that the change in the nutrition type was important for controlling enzyme activities in the organs examined and, consequently, for directing the pathway of ammonium nitrogen assimilation. As long as the plants remained heterotrophic, glutamine-dependent asparagine synthetase of cotyledons and glutamine synthetase of leaves apparently played a major role in the assimilation of ammonium nitrogen. In symbiotrophic plants, root nodules became an exclusive site of asparagine synthesis, and the role of leaf glutamine synthetase increased. Unlike glutamine synthetase and asparagine synthetase, glutamate dehydrogenase activity was present in all organs examined and was less dependent on the nutrition type. This was also indicated by a weak correlation of glutamate dehydrogenase activity with the dynamics of free amino acid and amide content in these organs. It is supposed that glutamine synthetase plays a leading role in both the primary assimilation of ammonium, produced during symbiotic fixation of molecular nitrogen in root nodules, and in its secondary assimilation in cotyledons and leaves. On the other hand, secondary nitrogen assimilation in the axial organs occurs via an alternative glutamate dehydrogenase pathway.     

Utilization of Nitrogen Sources by Immature Soybean Cotyledons in Culture

Immature Glycine max (L.) Merrill cotyledons were cultured ina defined medium containing different nitrogen sources. Glutaminewas the most efficient source in terms of protein accumulationin the cotyledons. Asparagine was less efficient (about 70 percent that of glutamine) while allantoin was a poor source ofnitrogen. This was also true for older cotyledons where asparaginaseand allantoinase activities were maximal. The utilization ofboth asparagine and allantoin (but not glutamine) was totallyinhibited by methionine sulfoximine suggesting that their metabolisminvolves ammonia assimilation via glutamine synthetase. Apparently,neither exogenous or endogenously-generated ammonia had mucheffect on glutamine utilization, but ammonia did have a smallinhibitory effect on asparagine, which may in part account forthe lower efficiency observed with this amide. Glycine max, soybean, cotyledon culture, nitrogen metabolism     

Distribution of {beta}-Thioglucosidase Activity in Intact Plants, Cell and Tissue 6Brassica napus L.

Distribution of myrosinase activity in extracts from seeds,intact plants, cell cultures and regenerated callus and plantsof Brassica napus L. was determined by the rate of glucose formationfrom glucosinolate hydrolysis. Calli with shoots and regeneratedplants were obtained from protoplasts or from explants. Of the seedling organs from Brassica napus L. cv. Niklas, hypocotylsshowed the highest myrosinase activity. In cotyledons a nearlyconstant enzyme activity was determined over the first 6 d,followed by a gradual decline. Roots showed a fast decline inenzyme activity over the investigated period. Freshly-isolated protoplasts contained less myrosinase activitythan the original intact tissue. The enzyme activity in developingcalli generally decreased during the first culture periods.After the initial decline a low activity was found which wasstable for a period of more than 2 years. The enzyme activityshowed fluctuations when measured at different times after mediumchange. Protoplast calli with regenerated shoots showed a considerablyhigher myrosinase activity than calli without shoots. Myrosinaseactivity was also found in explant calli including explant callifrom cotyledons and hypocotyls after induction of shoots. Myrosinase activity in seeds from 21 cultivars of Brassica napus,Brassica campestris, Sinapis alba and Raphanus sativus was testedand the highest myrosinase activity was found in seeds fromthe Sinapis alba cultivar Trico while the lowest activity wasfound in the Brassica campestris cultivar Rapido III. Leaf, stem and inflorescence from flowering regenerated or seed-grownplants contained a low but significant myrosinase activity.In contrast, roots showed a high myrosinase activity. The resultsobtained from regenerated plants indicate that the myrosinasesystem is stable in vitro culture, and that the glucosinolate-myrosinasesystem is active in calli tissue. Key words: Myrosinase (thioglucoside glucohydrolase, E.C. 3.2.3.1), in vitro cultures, intact plants