Effect Of Exogenous Amino Acids Research Articles

Foliar application of phenylalanine, tryptophan, and tyrosine in fragrant rice production: Aroma, yield, grain quality, and economic return

Phenylalanine, tryptophan, and tyrosine are amino acids that are important to the growth and development of crops. However, the application of these amino acids in fragrant rice (Oryza sativa L.) production has not been reported. The present study aimed to investigate the effects of exogenous amino acids on aroma, yield, grain quality, and economic return of fragrant rice. At the heading stage, phenylalanine, tryptophan, and tyrosine at 0.50, 1.00, and 2.00 g L−1 were foliar applied to fragrant rice plants respectively and the treatment sprayed with distilled water was taken as control. The results showed that the foliar application of these amino acids significantly increased grain yield and seed-setting rate of fragrant rice. The total return and net income of fragrant rice production increased due to amino acids and the highest or equally highest net income and benefit-cost ratio was recorded in phenylalanine treatments with 0.50 and 1.00 g L−1 and tryptophan with 1.00 g L−1. However, amino acids applied at 2.00 g L−1 significantly decreased the benefit-cost ratio. Foliar application of phenylalanine, tryptophan, and tyrosine significantly enhanced superoxide dismutase activity. Phenylalanine treatments also increased the chlorophyll content. Compared with control, phenylalanine and tryptophan treatments significantly increased the grain content of 2-acetyl-1-pyrroline (2-AP) which is the key volatile component of fragrant rice aroma. The increased 2-AP content was explained by enhanced activity of proline dehydrogenase which plays an important part in catalyzing the conversion from proline to 2-AP. In conclusion, foliar application of phenylalanine, tryptophan, and tyrosine improved yield formation and economic return of fragrant rice. Foliar application of phenylalanine and tryptophan also enhanced 2-AP biosynthesis in fragrant rice.

Effects of exogenous amino acids on the freeze-drying survival rate of Lactiplantibacillus plantarum LIP-1

How to improve the freeze-drying survival rate of Lactic acid bacteria (LAB) by increasing its resistance is a problem that is worth paying attention to. We evaluated the effect of exogenous amino acids on freeze-drying resistance of strain using Lactiplantibacillus plantarum LIP-1 as model probiotics. We found that the survival rate of L. plantarum LIP-1 after freeze-dried could be significantly enhanced by adding 0.04 g/L glycine to medium (p < 0.05), compare with other amino acids. Mechanistic studies showed that the content of peptidoglycan was increased to improve the integrity of the cell wall via metabolizing glycine; By metabolizing glycine, the content of unsaturated fatty acid was increased, such as C19cyc11, to promote membrane fluidity. In addition, the metabolism of glycine could neutralize the acidic intracellular environment to reduce DNA damage, thus improve the survival rate of freeze-dried strain. These findings may provide a new idea for industrialization of LAB powder.

Monitoring Role of Exogenous Amino Acids on the Proteinogenic and Ionic Responses of Lettuce Plants under Salinity Stress Conditions

Lettuce plants (Lactuca sativa L.) were grown under salinity stress conditions. Amino acids (histidine (His), lysine (Lys), phenylalanine (Phe), and threonine (Thr)) were individually applied to the seedlings to study their impact on the status of the photosynthetic pigments, ion absorption, proteinogenic metabolism, and peroxidase activity. Investigating the effect of exogenous amino acids on the metabolism processes showed their potential role in inducing salt stress tolerance in lettuce plants. Generally, a destructive impact on lettuce plant morphology was observed when the plants were exposed to salt stress. In contrast, the significant (p &lt; 0.05) mitigation of salt stress was registered when EAAs were applied to the stressed seedlings while using Threonine and lysine enhanced the status of the plants under salinity stress. For the salt treatment, the maximum electric conductivity (580.2 μS/g) was reported while applying EAAs to stressed plants’ decreased EC, and the data ranged from 522 to 554 μS/g. EAAs decreased the chloride ions in the leaves by 23–30% compared to in the stressed plants. Additionally, the sodium contents were mitigated when the stressed plants were sprayed with EAAs. In contrast, applying EAAs enhanced the potassium uptake, and Thr gave the highest K+ contents (3022 μg/g). EAAs increased the chlorophyll content compared to the control except when histidine was applied, while the carotene contents significantly increased when histidine and phenylalanine were used. Endogenous amino acids are highly expressed in non-stressed lettuce plants compared to the stressed ones. Under salt stress conditions, the threonine usage increased the expression of proteinogenic amino acids except methionine and tyrosine. Compared to the salt-stressed plants, the peroxidase activity significantly decreased in the other treatments, which fell by over 32% when His, Lys, and Phe were applied.

The effects of exogenous amino acids on production of microcystin variants in Microcystis aeruginosa

Dissolved free amino acids are a significant component of dissolved organic nitrogen (DON) in natural waterbodies. The effects of four amino acids (glutamic acid, phenylalanine, leucine, and arginine) on the growth and microcystins (MCs) production of Microcystis aeruginosa were studied in batch culture. The profiles of five MCs variants and the expression levels of target genes involved in MCs biosynthesis and nitrogen metabolism were measured. When amino acids were used as the sole nitrogen source instead of nitrate at different levels (0.5, 2.0 and 8.0 mg/L based on N) in BG-11 medium, algal cell growth and intracellular MCs quotas were inhibited slightly by the treatments with glutamic acid and arginine. The treatments with phenylalanine and leucine, on the other hand, had a strong inhibitory effect on algal cell growth and MCs production. Moreover, the concentrations of Chlorophyll a, phycocyanin and allophycocyanin in cells cultured in glutamic acid, leucine and phenylalanine were lower than those in the control group with nitrate as nitrogen source. The existence of leucine or phenylalanine can lead to a significant increase in the relative abundance of MCs variants structured with the corresponding amino acids. The expression of microcystin-producing gene mcyD was downregulated while the gene pipX associated with nitrogen metabolism was upregulated during the cultivation of M. aeruginosa with amino acids as sole nitrogen source. M. aeruginosa undergoes significant alterations due to exogenous amino acids and exhibits advanced strategies for MCs production.

Contribution of amino acids to Alicyclobacillus acidoterrestris DSM 3922T resistance towards acid stress

Spoilage of juice and beverages by a thermo-acidophilic bacterium, Alicyclobacillus acidoterrestris, has been considered to be a major and widespread concern for juice industry. Acid-resistant property of A. acidoterrestris supports its survival and multiplication in acidic juice and challenges the development of corresponding control measures. In this study, intracellular amino acid differences caused by acid stress (pH 3.0, 1 h) were determined by targeted metabolomics. The effect of exogenous amino acids on acid resistance of A. acidoterrestris and the related mechanisms were also investigated. The results showed that acid stress affected the amino acid metabolism of A. acidoterrestris, and the selected glutamate, arginine, and lysine contributed to its survival under acid stress. Exogenous glutamate, arginine, and lysine significantly increased the intracellular pH and ATP level, alleviated cell membrane damage, reduced surface roughness, and suppressed deformation caused by acid stress. Additionally, the up-regulated gadA and speA genes and the enhanced enzymatic activity confirmed that glutamate and arginine decarboxylase systems played a crucial role in maintaining pH homeostasis of A. acidoterrestris under acid stress. Our research reveals an important factor contributing to acid resistance of A. acidoterrestris, which provides an alternative target for effectively controlling this contaminant in fruit juices.

Translocation, Leaf Distribution, and Nutritional Status of Manganese in Cucumber Plants as Affected by Foliar Application of Exogenous Amino Acids

The present study was carried out to investigate the effect of exogenous amino acids (AAs) on leaf apoplastic retention and symplastic absorption, leaf distribution, translocation, and nutritional status of manganese (Mn) in cucumber plants. Results showed that application of AAs increased the symplastic to apoplastic Mn proportion in leaves of cucumber. Furthermore, a greater part of Mn applied with AAs accumulated in the internal area of newly growing leaves suggesting the transport of Mn mostly in the phloem in the presence of AAs. In contrast, Mn preferentially accumulated in the marginal area of treated leaves indicating the Mn movement in xylem according to the transpiration stream. The AAs used in the present study also increased the relative translocation of Mn from treated leaves to the upper leaves and roots. More time was needed for long-distance translocation of Mn from treated leaves to the upper leaves than that to the roots. Application of AAs mainly increased the transport of Mn in the phloem. The leaf PO (peroxidase) activity was the better marker to index the nutritional status of Mn than PPO (polyphenoloxidase) activity. In conclusion, exogenous AAs especially lysine (Lys) promoted the mobility and bioavailability of applied Mn as foliar spray.

Improvement of cold tolerance of winter oilseed rape by exogenous amino acids

Research was carried out at the Experimental Station of Aleksandras Stulginskis University on Calc(ar)i-Endohypogleyic Luvisol (LVg-n-w-cc) during 2014–2017. The effect of exogenous amino acids on the cold tolerance of the linear cultivar ‘Cult’ of winter oilseed rape (Brassica napus L. spp. oleifera biennis Metzg.) was studied. In autumn the plants were sprayed with L-proline and L-glutamic acid one or two times. The biometric parameters of winter oilseed rape prepared for wintering, the amount of endogenous proline and over-wintering of plants were evaluated. It was determined that exogenous L-proline and L-glutamic acid stimulated the growth of green mass of the winter rape surface, accumulation of dry matter and increase of the root neck diameter in the autumn period. The investigated concentrations of L-proline and L-glutamic acid stimulated a more intensive synthesis of endogenous proline in winter oilseed rape plants during the preparation for wintering. The highest percentage of over-wintering has been determined in the variants where in the autumn plants were sprayed two times with 30 mM l–1 L-proline or 1.5 M l–1 L-glutamic acid.

Exogenous glycine inhibits root elongation and reduces nitrate-N uptake in pak choi (Brassica campestris ssp. Chinensis L.).

Nitrogen (N) supply, including NO3--N and organic N in the form of amino acids can influence the morphological attributes of plants. For example, amino acids contribute to plant nutrition; however, the effects of exogenous amino acids on NO3--N uptake and root morphology have received little attention. In this study, we evaluated the effects of exogenous glycine (Gly) on root growth and NO3--N uptake in pak choi (Brassica campestris ssp. Chinensis L.). Addition of Gly to NO3--N agar medium or hydroponic solution significantly decreased pak choi seedling root length; these effects of Gly on root morphology were not attributed to the proportion of N supply derived from Gly. When pak choi seedlings were exposed to mixtures of Gly and NO3--N in hydroponic culture, Gly significantly reduced 15NO3--N uptake but significantly increased the number of root tips per unit root length, root activity and 15NO3--N uptake rate per unit root length. In addition, 15N-Gly was taken up into the plants. In contrast to absorbed NO3--N, which was mostly transported to the shoots, a larger proportion of absorbed Gly was retained in the roots. Exogenous Gly enhanced root 1-aminocyclopropane-1-carboxylic acid synthase (ACS) and oxidase (ACO) activities and ethylene production. The ethylene antagonists aminoethoxyvinylglycine (0.5 μM AVG) and silver nitrate (10 μM AgNO3) partly reversed Gly-induced inhibition of primary root elongation on agar plates and increased the NO3--N uptake rate under hydroponic conditions, indicating exogenous Gly exerts these effects at least partly by enhancing ethylene production in roots. These findings suggest Gly substantially affects root morphology and N uptake and provide new information on the specific responses elicited by organic N sources.

Root uptake and xylem transport of cadmium in wheat and triticale as affected by exogenous amino acids

Organic acids exuded from plant roots significantly modify uptake and long-distance translocation of metals. Little is known about the effect of amino acids on metal ion uptake by plant roots. The present study investigated the effects of exogenous amino acids (histidine and glycine) in a nutrient solution on root uptake and xylem sap transport of cadmium (Cd) in triticale (× Triticosecale cv. Elinor) and bread wheat (Triticum aestivum L. cv. Back Cross Rushan). Plant seedlings were grown in a Cd-free modified Hoagland nutrient solution to which 1 µm Cd was added with either 50 µm histidine or 50 µm glycine or without amino acids at 4 weeks after germination. A control treatment consisted of a nutrient solution free of Cd and amino acids. In bread wheat, addition of histidine to the Cd-containing nutrient solution resulted in a higher operationally defined symplastic Cd fraction but a lower apoplastic one in the roots. In triticale, addition of either amino acid decreased the symplastic Cd fraction but increased the apoplastic one. Addition of histidine to the nutrient solution increased Cd concentration in wheat xylem sap but had no significant effect on Cd concentration in triticale xylem sap. Compared with the Cd-only treatment, the glycine-containing treatment led to significantly reduced Cd concentrations in xylem sap of both plant species. Wheat plants supplied with histidine and Cd accumulated greater amounts of Cd in their shoots than those supplied with Cd alone. Glycine had no significant effects on the Cd content of wheat shoots but decreased it in triticale shoots. Results indicate that the effects of amino acids on plant root uptake and xylem sap translocation of Cd depend on the type of amino acid supplemented. This finding is of great importance for selecting and/or breeding cultivars with Cd-toxicity tolerance.

Symplastic and apoplastic uptake and root to shoot translocation of nickel in wheat as affected by exogenous amino acids

This study investigated the effect of exogenous amino acids on apoplastic and symplastic uptake and root to shoot translocation of nickel (Ni) in two wheat cultivars. Seedlings of a bread (Triticum aestivum cv. Back Cross) and a durum wheat cultivar (T. durum cv. Durum) were grown in a modified Johnson nutrient solution and exposed to two levels (50 and 100μM) of histidine, glycine, and glutamine. Application of amino acids resulted in increasing symplastic to apoplastic Ni ratio in roots of both wheat cultivars, although glutamine and glycine were more effective than histidine under our experimental conditions. The amino acid used in the present study generally increased the relative transport of Ni from the roots to shoots in both wheat cultivars. Higher amounts of Ni were translocated to wheat shoots in the presence of histidine than the other amino acids studied, which indicated that histidine was more effective in translocation of Ni from roots to shoots. Amino acids used in the present study largely increased root symplastic Ni, but shoot Ni accumulation was much lower than the total Ni accumulation in roots, indicating a large proportion of Ni was retained or immobilized in wheat roots (either in the apoplastic or symplastic space), with only a very small fraction of Ni being translocated from the root to the shoot. According to the results, glutamine and glycine were more effective than histidine in enhancing the symplastic to apoplastic Ni ratio in the roots, while more Ni was translocated from the roots to the shoots in the presence of histidine.

Effect of exogenous amino acids on Cu uptake and translocation in maize seedlings

This study aimed to assess the effects of four contrasting proteinogenic amino acids on copper (Cu) uptake and translocation in maize (Zea mays L.) seedlings grown in a modified Hoagland solution. Glycine, aspartic acid and lysine at three concentrations (10, 25 and 100 μM) did not have any significant effect on Cu uptake and translocation in maize seedlings over a two-day experimental period. However, cysteine (a reductive amino acid) at the three concentrations increased very significantly (P < 0.01) Cu accumulations in the root symplast and the shoots of maize seedlings in comparison to the control. Cu uptake in the whole plant and Cu translocation from root to shoot were also increased in the cysteine treatments. In the 25 μM cysteine treatment, where cysteine was in moderate excess, the Cu uptake in the whole plant and Cu translocation from root to shoot were significantly (P < 0.01) higher than those of the 10 or 100 μM cysteine treatments, where the concentration of cysteine was equivalent to that of Cu(II) or in great excess according to the stoichiometry of the redox reaction of cysteine with Cu(II). It is hypothesized that the cysteine-induced oxidation state alteration from Cu(II) to Cu(I) could be responsible for the increased Cu uptake and Cu translocation, on the ground that Cu(I), as free cuprous ion or cysteine cuprous complex, may be more available to maize roots than Cu(II).

The effects of exogenous amino acids on the relaxant responses of pig urethral smooth muscle evoked by stimulation of the inhibitory nitrergic nerves

Inhibitory innervation of urethral smooth muscle is mediated partly through release of NO. We investigated the mechanisms involved in the supply of the substrate L: -arginine to NO synthase by examining the relaxant response of the muscle to electrical field stimulation (EFS) and the effects of addition of amino acids to the bathing medium. Relaxant responses persisted during hours of repetitive stimulation but were enhanced rapidly by addition of L: -arginine (the "arginine paradox"). Addition of L: -lysine (competes with L: -arginine for transport on the y(+) carrier) and L: -glutamine (competing on the y(+)L carrier) attenuated the enhancement. Enhancement persisted after washing but was reversed by application of L: -lysine, suggesting that exogenous L: -arginine fills an intracellular pool and that L: -lysine can trans-stimulate its efflux from the pool. After prolonged depolarization in high-K(+), Na(+)-free solution the relaxant response became purely nitrergic. Addition of L: -arginine during the exposure continued to enhance the subsequent responses but L: -glutamine added with L: -arginine, could no longer reduce this enhancement. The results show the arginine paradox in inhibitory nerves and suggest the involvement of y(+) and y(+)L carriers in the transport of L: -arginine.

An abundant supply of amino acids enhances the metabolic effect of exercise on muscle protein.

Six normal untrained men were studied during the intravenous infusion of a balanced amino acid mixture (approximately 0.15 g.kg-1.h-1 for 3 h) at rest and after a leg resistance exercise routine to test the influence of exercise on the regulation of muscle protein kinetics by hyperaminoacidemia. Leg muscle protein kinetics and transport of selected amino acids (alanine, phenylalanine, leucine, and lysine) were isotopically determined using a model based on arteriovenous blood samples and muscle biopsy. The intravenous amino acid infusion resulted in comparable increases in arterial amino acid concentrations at rest and after exercise, whereas leg blood flow was 64 +/- 5% greater after exercise than at rest. During hyperaminoacidemia, the increases in amino acid transport above basal were 30-100% greater after exercise than at rest. Increases in muscle protein synthesis were also greater after exercise than at rest (291 +/- 42% vs. 141 +/- 45%). Muscle protein breakdown was not significantly affected by hyperminoacidemia either at rest or after exercise. We conclude that the stimulatory effect of exogenous amino acids on muscle protein synthesis is enhanced by prior exercise, perhaps in part because of enhanced blood flow. Our results imply that protein intake immediately after exercise may be more anabolic than when ingested at some later time.

Effect of exogenous amino acids, glucose and citric acid on the patterns of short-term accumulation and loss of amino acids in the root-zone of sand-cultured forage rape (Brassica napus L.)

Amino acid loss from the roots of 25-day-old, sterile and non-sterile sand-grown forage rape plants, was determined over periods of up to 3.5 hours. Amino acid accumulation in the root-zone of sterile plants was concentration-dependent giving a convex accumulation profile. Amino acid levels in the root zone of non-sterile plants rapidly attained steady state values. Microbial assimilation of amino acids within the root zone appeared to lower amino acid concentrations, resulting in an underestimation of rates of amino acid loss from roots. The concentrations of most amino acids were higher after selected amino acids were supplied to the root zone. The response to exogenous acids was dependent on the concentration and composition of the acids added. Addition of a mixture containing ASN, GLN and GABA, each at 0.25 mM resulted in a greater increase in individual and total acid levels compared with a mixture containing ALA, SER, GLY and THR at the same concentration. Apparently, amino acids supplied exogenously competed with acids lost from the plant, by providing an alternative nutrient source for root zone micro-organisms. Addition of glucose and citric acid had a similar effect to addition of ALA, SER, GLY and THR, but were less effective than ASN, GLN and GABA at all concentrations tested. The nitrogen-rich amino acids ASN and GLN, and the γ-amino acid, GABA, appeared to compete more effectively with plant-derived acids than did ALA, SER, GLY and THR, the most abundent constituents of the plant-derived acids, which had the highest calculated rates of microbial consumption. Therefore, although bacterial consumption showed a dependence on amino acid concentration, a degree of selectivity for nitrogen-rich acids and gaba was also apparent.

Effect of exogenous amino acids on the intracellular content of proline and other amino acids in Daucus carota cells

The effect of tryptophan on the biosynthesis of proline has been investigated. Cells of Daucus carota grown in B5 medium supplemented with 5×10(-4)M tryptophan acquired the ability to grow in the presence of inhibitory concentrations of azetidine-2-carboxylic acid, an analog of proline. When trp was added to carrot cell cultures at sub-growth inhibiting concentrations, overproduction of intracellular free proline was observed. An increase was also observed for lys, his, ala, leu and phe. Likewise, the addition of asparagine, glutamic acid and phenylalanine to the medium stimulated the intracellular increase of free proline and other amino acids.

Effects of exogenous amino acids on the multiplication of porcine Escherichia coli

The multiplication rates of 70 porcine Escherichia coli strains were compared in minimal medium and in medium supplemented with aspartic acid, lysine, serine and threonine, which were the amino acids taken up during multiplication of porcine E. coli in a complex medium. The effects of these amino acids singly or in combinations and the amino acids norleucine and norvaline on the growth of porcine E. coli were studied. Together, aspartic acid, threonine, serine and lysine increased the multiplication rates of 42.9% of the strains, an effect traced to aspartic acid, but they had no effect on an equal number of strains. The rest were inhibited, and this effect was traced to serine. Cysteine, threonine, leucine and phenylalanine singly inhibited some or all of the strains tested. Norleucine and to a lesser extent, norvaline greatly prolonged the lag phase of culture in minimal medium. The inhibitory effect of norleucine was reversed by only methionine, although isoleucine, leucine and valine which were more effective in norvaline inhibition, also showed limited antagonism to norleucine inhibition.

Effects of Exogenous Amino Acids on Iron Uptake in Relation to their Effects on Photoperiodic Flowering in &lt;italic&gt;Lemna paucicostata&lt;/italic&gt; 6746

Effects of Exogenous Amino Acids on Iron Uptake in Relation to their Effects on Photoperiodic Flowering in &lt;italic&gt;Lemna paucicostata&lt;/italic&gt; 6746

Effects of exogenous amino acids on growth and activity of four aspartate pathway enzymes in barley

Excised barley embryos were grown in the presence of 1 mM lysine, threonine, methionine and isoleucine, alone and in combinations. Growth was similar in all treatments except lysine plus threonine, where growth was severely inhibited. Activities of four regulatory biosynthetic enzymes were measured and expressed on a protein or fresh weight basis to assess possible repression/derepression under these conditions. Aspartate kinase (EC 2.7.2.4) (AK) activity and sensitivity to feedback regulators did not vary greatly between treatments. The activity and feedback sensitivity of homoserine dehydrogenase (EC 1.1.1.3) (HSDH) also showed little variation. Cystathionine synthase (EC 4.2.99.x) (CS) activity was markedly reduced in plants grown in the presence of methionine, and increased nearly 4-fold in the presence of lysine plus threonine, a condition in which methionine is limiting. Activity increased to a lesser extent in plants grown in the presence of threonine alone. Threonine synthase (EC 4.2.99.2) (TS) activity in the seedlings was reduced by up to one half in the presence of methionine, and to a smaller degree in the presence of isoleucine. None of the treatments led to increased activity of this enzyme.

Effect of exogenous amino acids on the contractility and nitrogenous metabolism of anoxic heart.

The effect of exogenous glutamic acid and arginine on the contractility of isolated perfused rat heart and on the metabolism of some nitrogenous compounds was studied. Sixty-minute anoxic perfusion (95% N2 + 5% CO2) led to a fall in developed isovolumic pressure and an elevation in diastolic pressure, to an increase in the production of alanine, glutamine, and ammonia, and to a decrease in the tissue content of aspartate and glutamate. The total pool of free amino acids and taurine under these conditions remained unchanged. Subsequent 40-min reoxygenation partially restored the contractile function. Addition of 3.5 mM glutamic acid or 5 mM arginine into the perfusate before anoxia resulted in a higher level of developed pressure and a lower level of diastolic pressure during anoxia and almost complete recovery of cardiac function after subsequent reoxygenation. Both amino acids had no effect on ammonia formation by the anoxic heart but enhanced its binding in myocardial tissue via formation of glutamine and urea. It is suggested that the exogenous amino acid effect on anoxic heart is mediated by activation of substrate phosphorylation rather than the ability to bind tissue ammonia.

Effects of Exogenous Amino Acids on in Androgenesis of Datura

Summary A study was carried out on the effects of 17 L-amino acids incorporated into an optimal medium, with and without inorganic nitrogen, on the androgenesis of cultured anthers of Datura metel. L. In the presence of inorganic nitrogen, certain amino acids such as glutamine, glutamic acid, proline and γ-amino butyric acid (GABA) and to a much lesser degree aspartic acid, increase considerably the percentage of embryogenic anthers and microspores. The other amino acids tested were either without any apparent action (serine, agrinine and cystine) or inhibited androgenesis partially or fully (histidine, threonine, glycine, valine, leucine, lysine, alanine, tyrosine and phenylalanine). In the absence of inorganic nitrogen (nitrate and ammonium ions) androgenic embyros were formed only in the presence of glutamine, GABA or glutamic acid but the percentages were very low; and most of those embryos showed anomalies or teratologic structures. Our study thus demonstrated the absolute need for an inorganic nitrogen source in addition to those amino acids whose effect was positive, on the optimal development of microspore-derived embryogenesis in vitro (androgenesis).