Biochanin A improves fibre fermentation by cellulolytic bacteria

Aims

The objective was to determine the effect of the isoflavone biochanin A (BCA) on rumen cellulolytic bacteria and consequent fermentative activity.

Methods and Results

When bovine microbial rumen cell suspensions (n = 3) were incubated (24 h, 39°C) with ground hay, cellulolytic bacteria proliferated, short‐chain fatty acids were produced and pH declined. BCA (30 μg ml^?1) had no effect on the number of cellulolytic bacteria or pH, but increased acetate, propionate and total SCFA production. Addition of BCA improved total digestibility when cell suspensions (n = 3) were incubated (48 h, 39°C) with ground hay, Avicel, or filter paper. Fibrobacter succinogenes S85, Ruminococcus flavefaciens 8 and Ruminococcus albus 8 were directly inhibited by BCA. Synergistic antimicrobial activity was observed with BCA and heat killed cultures of cellulolytic bacteria, but the effects were species dependent.

Conclusions

These results indicate that BCA improves fibre degradation by influencing cellulolytic bacteria competition and guild composition.

Significance and Impact of the Study

BCA could serve as a feed additive to improve cellulosis when cattle are consuming high‐fibre diets. Future research is needed to evaluate the effect of BCA on fibre degradation and utilization in vivo.     

The characterization of lactic acid producing bacteria from the rumen of dairy cattle grazing on improved pasture supplemented with wheat and barley grain

Aims:  To identify and characterize the major lactic acid bacteria in the rumen of dairy cattle grazing improved pasture of rye grass and white clover and receiving a maize silage and grain supplement with and without virginiamycin.
Methods and Results:  Eighty-five bacterial isolates were obtained from the rumen of 16 Holstein-Friesian dairy cows. The isolates were initially grouped on the basis of their Gram morphology and by restriction fragment length polymorphism analysis of the PCR amplified 16S rDNA. A more definitive analysis was undertaken by comparing the 16S rDNA sequences. Many of the isolates were closely related to other previously characterized rumen bacteria, including Streptococcus bovis, Lactobacillus vitulinus , Butyrivibrio fibrisolvens , Prevotella bryantii and Selenomonas ruminantium . The in vitro production of l - and/or d -lactate was seen with all but five of the isolates examined, many of which were also resistant to virginiamycin.
Conclusion:  Supplementation of grain with virginiamycin may reduce the risk of acidosis but does not prevent its occurrence in dairy cattle grazing improved pasture.
Significance and Impact of the Study:  This study shows that lactic acid production is caused, not only by various thoroughly researched types of bacteria, but also by others previously identified in the rumen but not further characterized.     

Effect of glyphosate contaminated feed on rumen fermentation parameters and in sacco degradation of grass hay and corn grain

Four rumen fistulated wethers were used to investigate the effect of glyphosate contaminated feed on rumen fermentation. The rations were based on corn silage, urea and a vitamin-mineral premix, either in the absence or presence of 0.77?g glyphosate per kg DM. Furthermore, rations were fed either with or without aromatic amino acid supplementation. During four periods of 28 days, sheep received each of the four dietary treatments according to a Latin square. After 14 days of adaptation rumen fermentation parameters (pH, ammonia, volatile fatty acids) were measured on day 15 over a five-hour period after the morning feeding. The remaining 13 days served for in sacco degradation studies with grass hay and corn grain. Ammonia (NH₃) and pH of rumen fluid were within the normal range for all dietary treatments (NH₃: 9.1 – 32.3?mmol·l^???1, pH: 6.2 – 6.7). Neither rumen fermentation parameters nor in sacco DM and NDF degradation of incubated feedstuffs were significantly affected by glyphosate, with or without aromatic amino acid supplementation. Kinetic profiles of the in sacco dry matter and NDF degradation of grass hay were almost identical for the dietary treatments.     

Effect of arbuscular mycorrhizal fungi on phytoextraction by corn (Zea mays) of lead-contaminated soil

The role of arbuscular mycorrhizal fungi (AMF) in lead (Pb) uptake by corn (Zea mays) grown in soil supplemented with Pb was examined. Plants were subjected to four Pb levels: 0 (control); 10 (low); 100 (medium); and 500 mg L(-1) (high). At each Pb level, plants were grown in soil without and with fungicide (benomyl) (20 mg kg(-1)) to suppress AMF activity. Benomyl significantly reduced AMF colonization at high. medium, and zero Pb exposures. Benomyl application resulted in significantly lower concentrations of phosphorus in leaves at low and medium Pb exposures. The benomyl-treated plants had higher Pb and manganese concentrations in leaves than plants not treated with benomyl. In addition, benomyl-treated plants had generally lower concentrations of zinc and copper in leaves than plants not treated with benomyl. These results suggest that the role of AMF in heavy metal uptake is metal specific. Based on this work, the use of benomyl on soils contaminated with Pb can be recommended in phytoextraction.     

Degradation of the isoflavone biochanin a by isolates of Nectria haematococca (Fusarium solani)

Twelve isolates of Nectria haematococca, mating population VI (Fusarium solani) previously characterized for their virulence on pea plants and their ability to degrade the phytoalexin pisatin were assayed for the catabolism of the isoflavone biochanin A (5,7-dihydroxy-4′-methoxyisoflavone). Eleven isolates catabolized the isoflavone along the pathway: biochanin A → dihydrobiochanin A → 3-(p-methoxyphenyl)-6-hydroxy-γ-pyrone → p-methoxyphenylacetic acid → p-hydroxyphenylacetic acid → 3,4-dihydroxyphenylacetic acid.     

Effect of different levels of concentrate on ruminal microorganisms and rumen fermentation in Nellore steers

The aim of this study was to investigate the effect of different dietary levels of concentrate on feed intake, digestibility, ruminal fermentation and microbial population in steers. Eight Nellore steers fitted with ruminal cannulas were used in a double 4 × 4 Latin square design experiment. The dietary treatments consist of four different proportions of concentrate to roughage: 30:70, 40:60, 60:40 and 80:20% in the dry matter, resulting in Diets 30, 40, 60 and 80, respectively. The roughage was corn silage, and the concentrate was composed of corn, soybean meal and urea. Apparent digestibility of organic matter and crude protein showed a linear association with concentrate proportion (p = 0.01), but the increased concentrate levels did not affect the digestibility of fibre. The lowest ruminal pH-values were observed in animals fed with Diet 80, remaining below pH 6.0 from 6 h after feeding, while in the other diets, the ruminal pH was below 6.0 not before 12 h after feeding. After feeding Diet 80, the ammonia concentration in the rumen was significantly the highest. Higher dietary concentrate levels resulted in a linear increase of propionic acid concentrations, a linear reduction of the ratio acetic acid to propionic acid (p < 0.01) and a linear increased synthesis of microbial nitrogen (p < 0.001). The predicted production of methane was lower in diets with greater amounts of concentrate (p = 0.032). The population of methanogens, R. flavefaciens and R. albus decreased with higher concentrate levels, while the population of S. ruminantium increased (p < 0.05). The results indicate that greater amounts of concentrate do not decrease ruminal pH-values as much as expected and inhibit some cellulolytic bacteria without impairing the dry matter intake and fibre digestibility in Nellore steers.     

Effect of a blend of essential oil compounds on the colonization of starch-rich substrates by bacteria in the rumen

AIMS: To investigate the mode of action of a blend of essential oil compounds on the colonization of starch-rich substrates by rumen bacteria. METHODS AND RESULTS: Starch-rich substrates were incubated, in nylon bags, in the rumen of sheep organized in a 4 x 4 latin square design and receiving a 60:40 silage : concentrate diet. The concentrate was either high or low in crude protein, and the diet was supplemented or not with a commercial blend of essential oil compounds (110 mg per day). The total genomic DNA was extracted from the residues in the bags. The total eubacterial DNA was quantified by real-time PCR and the proportion of Ruminobacter amylophilus, Streptococcus bovis and Prevotella bryantii was determined. Neither the supplementation with essential oil compounds nor the amount of crude protein affected the colonization of the substrates by the bacteria quantified. However, colonization was significantly affected by the substrate colonized. CONCLUSIONS: The effect of essential oils on the colonization of starch-rich substrates is not mediated through the selective inhibition of R. amylophilus. SIGNIFICANCE AND IMPACT OF THE STUDY: This study enhances our understanding of the colonization of starch-rich substrates, as well as of the mode of action of the essential oils as rumen manipulating agents.     

Effect of a controlled-release urea supplement on rumen fermentation in sheep fed a diet of sugar cane tops (Saccharum officinarum), corn stubble (Zea mays) and King grass (Pennisetum purpureum)

Four cannulated sheep were used to study ruminal fermentation of a diet consisting of 60% sugar cane tops (Saccharum officinarum), 30% corn stubble (Zea mays), 10% King grass (Pennisetum purpureum) and 0% (control), 10, 20 or 30% controlled-release urea supplement (CRUS) (diets 1, 2, 3 and 4, respectively). Average ruminal pH did not differ among diets (P>0.05), but during the first 6 h of sampling tended to be higher for CRUS diets. Ammonia concentrations were higher (P<0.01) in all treatments over controls, indicating microbial protein generation. Acetic acid production (mM/1) decreased (P<0.05), propionic acid increased (P<0.05), while butyric acid production did not differ among CRUS diets and controls (P>0.05). Total amounts of ruminal VFA were lowest (P<0.01) in controls, while CRUS diets produced more of these energy sources. Supplementation of the high fiber diets with 10, 20 or 30% CRUS increasingly improved rumen fermentation, ammonia supply and VFA production. The results show that low quality forages (up to 70% DMI) can be used efficiently by sheep when conditions for ruminal microorganism are improved with a controlled-release urea supplement.     

Effect of zinc and phosphorus on the grain yield of corn (Zea mays L.) grown on a calcareous soil

Summary In a field experiment, more than 22% increase in the grain yield of corn was obtained by the application of 50 kg ZnSO₄/ha. Grain yields were also increased by increasing the level of applied phosphorus. Positive relationship was obtained between Zn and P, the phosphorus treatment increased Zn uptake by grains and vice versa.     

Effect of grain type and processing method on rumen fermentation and milk rumenic acid production

It was hypothesized that differences in starch degradability account for observed differences in rumen vaccenic acid (t11-18:1) and milk rumenic acid (RA) concentrations. To test this hypothesis, starch degradability was varied through grain source and by processing. Eight Holstein cows in mid-lactation were assigned to two 4 × 4 Latin squares with four 21-day periods and four diets: dry rolled barley, ground barley, dry rolled corn and ground corn. Diets contained similar starch content and were supplemented with whole sunflower seed to provide similar total polyunsaturated fatty acid (PUFA) (18:2n-6 + 18:3n-3) contents. Forage/concentrate ratios of all diets were 42 : 58. Rumen, plasma and milk samples were collected in the third week of each period. In situ degradation rates (%/h) for rolled corn, ground corn, rolled barley and ground barley were 5.4, 8.9, 17.0 and 19.4, respectively, for dry matter (DM) and 6.3, 10.8, 25.3 and 43.8, respectively, for starch. DM intakes were greater for corn-based diets (CBD) than for barley-based diets (BBD) with no difference between rolled and ground diets. Daily minimum rumen pH was less (5.2 v. 5.5) and pH duration <5.8 (h/d) was greater (7.4 v. 4.3) for BBD than for CBD. Milk fat content and yield were less for BBD than for CBD with greater values observed for rolling compared with grinding. Variability in milk fat yield was strongly related (R2 = 0.55; P < 0.01) to total starch intake (45%) and milk c9t11-CLA (10%) and none of the t-18:1 isomers or CLA isomers that are typically associated with milk fat depression entered the model. The concentrations (%) of t10-18:1 and t11-18:1 were greater for BBD than for CBD in rumen contents (t10-18:1, 3.5 v. 1.3; t11-18:1, 3.2 v. 1.9), plasma (t10-18:1, 1.2 v. 0.2; t11-18:1, 0.97 v. 0.58) and milk (t10-18:1, 3.8 v. 1.0; t11-18:1, 2.6 v. 1.7) despite greater total PUFA intakes for CBD. Milk RA concentration was greater for BBD than for CBD (1.46 v. 0.89) but was not influenced by the method of grain processing. This study clearly demonstrated that the milk content and profile of t-18:1 and CLA isomers were more strongly influenced by the source of grain starch (barley > corn) than by the method of grain processing indicating that factors inherent in the source of starch were responsible for the observed differences and these factors could not be modified by the processing methods used in this study.     

Effect of zeolite A on rumen fermentation and phosphorus metabolism in dairy cows

The aim of the present study was to determine the effect of zeolite A on several physiological parameters and on mineral metabolism in the rumino-intestinal-tract of cows. Eight double fistulated (rumen and proximal duodenum) cows were fed maize silage, grass silage and concentrate. Zeolite A was added to the ration over a period of three weeks at 0, 10 and 20 g/kg dry matter (DM). The daily feed amounts were adjusted to the current performance and varied between 3.9 and 15.5 kg/d. Rumen fluid, duodenal chyme and faeces were sampled to characterise the nutrient digestibility. Blood samples were taken to analyse the concentration of inorganic phosphate. Zeolite A supplementation led to a significantly reduced ruminal DM digestibility and fermentation of organic matter. The molar proportion of acetate in the rumen increased, and propionate as well as valerate decreased significantly after zeolite A supplementation. The concentration of the total fatty acids and ruminal pH were not affected. No effect on faecal digestion of DM, organic matter nor on calcium and magnesium digestion was observed. Otherwise the phosphorus (P) concentration in rumen fluid correlated negatively with the mean zeolite A intake (r ² = 0.75; p = 0.0003). Further, the faecal excretion of P increased significantly for cows with the highest zeolite A dosage (36.9 g P/d) compared to the control group (29.9 g P/d). The lower digestibility of P resulted in a significantly decreased concentration of inorganic P in serum from a basal value of 2.05–1.16 mmol/l six days after starting zeolite A supplementation. The zeolite A treated cows showed a significantly higher Al concentration already in rumen fluid (14.31 and 13.84 mmol/l) compared to the control cows (6.33 mmol/l). The Al flow in the duodenum was also higher for zeolite A treated cows.     

模拟酸雨对不同类型玉米种子萌发和幼苗生长的影响

以普通玉米、糯玉米、爆裂玉米和甜玉米为材料,研究了不同模拟酸雨(pH 6.0、5.0、4.0、3.0、2.0、1.0)对玉米种子萌发和幼苗生长的影响.结果表明:pH 2.0 ～5.0模拟酸雨对玉米种子萌发和幼苗生长没有显著影响;pH l.0处理的普通玉米、糯玉米、爆裂玉米和甜玉米的种子发芽率分别为91.3％、68.7％、27.5％和11.7％.与pH 6.0处理(CK)相比,pH l.0模拟酸雨显著降低了玉米种子的发芽率、发芽指数、活力指数、发芽速度、苗高、根长、苗和根干物质、贮藏物质运转效率,延长了平均发芽时间.pH l.0模拟酸雨对玉米幼苗生长阶段的影响大于发芽阶段,对幼苗地下部分的影响大于地上部分;受基因型的影响,普通玉米和糯玉米的抗酸雨能力最强,其次为爆裂玉米,甜玉米最差.玉米属于抗酸雨作物,酸雨抑制阈值介于pH1.0～2.0之间.酸雨地区可优先选择种植普通玉米和糯玉米.     

Effect of whole-plant corn silage treated with lignocellulose-degrading bacteria on growth performance,rumen fermentation,and rumen microflora in sheep

Lignification of cellulose limits the effective utilisation of fibre in plant cell wall. Lignocellulose-degrading bacteria secrete enzymes that decompose lignin and have the potential to improve fibre digestibility. Therefore, this study aimed to investigate the effect of whole-plant corn silage inoculated with lignocellulose-degrading bacteria on the growth performance, rumen fermentation, and rumen microbiome in sheep. Twelve 2-month-old male hybrid sheep (Dorper ♂ × small-tailed Han ♀) were randomly assigned into two dietary groups (n = 6): (1) untreated whole-plant corn silage (WPCS) and (2) WPCS inoculated with bacterial inoculant (WPCSB). Whole-plant corn silage inoculated with bacterial inoculant had higher in situ NDF digestibility than WPCS. Sheep in the WPCSB group had significantly higher average daily gain, DM intake, and feed conversion rate than those in the WPCS group (P < 0.05). Furthermore, higher volatile fatty acid concentrations were detected in WPCSB rumen samples, leading to lower ruminal pH (P < 0.05). The WPCSB group showed higher abundance of Bacteroidetes and lower abundance of Firmicutes in the rumen microbiome than the WPCS group (P < 0.05). Multiple differential genera were identified, with Prevotella being the most dominant genus and more abundant in WPCSB samples. Moreover, the enriched functional attributes, including those associated with glycolysis/gluconeogenesis and citrate cycle, were more actively expressed in the WPCSB samples than in the WPCS samples. Additionally, certain glucoside hydrolases that hydrolyse the side chains of hemicelluloses and pectins were also actively expressed in the WPCSB microbiome. These findings suggested that WPCSB increased NDF digestibility in three ways: (1) by increasing the relative abundance of the most abundant genera, (2) by recruiting more functional features involved in glycolysis/gluconeogenesis and citrate cycle pathways, and (3) by increasing the relative abundance and/or expression activity of the glucoside hydrolases involved in hemicellulose and pectin metabolism. Our findings provide novel insights into the microbial mechanisms underlying improvement in the growth performance of sheep/ruminants. However, the biological mechanisms cannot be fully elucidated using only metagenomics tools; therefore, a combined multi-omics approach will be used in subsequent studies.     

Effects of hops (Humulus lupulus L.) extract on volatile fatty acid production by rumen bacteria

Aims: To determine the effects of hops extract on in vitro volatile fatty acid (VFA) production by bovine rumen micro‐organisms. Methods and Results: When mixed rumen microbes were suspended in media containing carbohydrates, the initial rates of VFA production were suppressed by β‐acid‐rich hops extract. The rates of VFA production increased over extended incubations (24 h), and hops extract caused an increase in the propionate to acetate ratio. Hops extract inhibited the growth and metabolism of Streptococcus bovis, but Selenomonas ruminantium and Megasphaera elsdenii were not affected. Likewise, the propionate production of M. elsdenii/S. bovis co‐cultures, but not M. elsdenii/S. ruminantium co‐cultures, was decreased in the presence of hops extract. Conclusions: These results are consistent with the hypothesis that the hops inhibit Gram‐positive lactic acid bacteria (S. bovis), and the rumen microbial community requires a period of adaptation before normal VFA production resumes. Selenomonas bovis and S. ruminantium both produce lactate, which is the substrate for propionate production by M. elsdenii. However, S. ruminantium has an outer membrane, while S. bovis does not. Significance and Impact of Study: The enhanced production of the gluconeogenesis precursor, propionic acid, provides further evidence that plant secondary metabolites from hops could be used to improve rumen fermentation.     

Effect of fibrolytic enzymes added to a Andropogon gayanus grass silage-concentrate diet on rumen fermentation in batch cultures and the artificial rumen (Rusitec)

In vitro batch cultures were used to screen four fibrolytic enzyme mixtures at two dosages added to a 60 : 40 silage : concentrate diet containing the C₄ tropical grass Andropogon gayanus grass ensiled at two maturities – vegetative stage (VS) and flowering stage (FS). Based on these studies, one enzyme mixture was selected to treat the same diets and evaluate its impact on fermentation using an artificial rumen (Rusitec). In vitro batch cultures were conducted as a completely randomized design with two runs, four replicates per run and 12 treatments in a factorial arrangement (four enzyme mixtures×three doses). Enzyme additives (E1, E2, E3 and E4) were commercial products and contained a range of endoglucanase, exoglucanase and xylanase activities. Enzymes were added to the complete diet 2 h before incubation at 0, 2 and 4 μl/g of dry matter (DM). Gas production (GP) was measured after 3, 6, 12, 24 and 48 h of incubation. Disappearance of DM (DMD), NDF (NDFD) and ADF (ADFD) were determined after 24 and 48 h. For all four enzyme mixtures, a dosage effect (P<0.05) was observed for NDFD and ADFD after 24 h and for DMD, NDFD and ADFD after 48 h of incubation of the VS diet. For the FS diet, a dosage effect was observed for GP and NDFD after 24 h and for GP, DMD, NDFD and ADFD after 48 h of incubation. There was no difference among enzyme mixtures nor was there an enzyme×dose interaction for the studied parameters. Because of the greatest numerical effect on NDF disappearance and the least cost price, enzyme mixture E2 at 4 µl/g of diet DM was selected for the Rusitec experiment. The enzyme did not impact (P>0.05) DM, N, NDF or ADF disappearance after 48 h of incubation nor daily ammonia-N, volatile fatty acids or CH₄ production. However, enzyme application increased (P<0.05) microbial N production in feed particle-associated (loosely-associated) and silage feed particle-bound (firmly associated) fractions. With A. gayanus silage diets, degradation may not be limited by microbial colonization, but rather by the ability of fibrolytic enzymes to degrade plant cell walls within this recalcitrant forage.     

Isolation and characterization of a ferulic acid esterase (Fae1A) from the rumen fungus Anaeromyces mucronatus

Aims: A novel ferulic acid esterase gene from rumen fungus Anaeromyces mucronatus was cloned, heteroexpressed in Escherichia coli and characterized. Methods and Results: A total of 30 clones exhibiting activity on α‐naphthyl acetate (α‐NA) were isolated from an A. mucronatus YE505 cDNA library. Sequence analysis revealed that these clones represented two esterase‐coding sequences. The gene, fae1A, showed highest amino acid sequence identity to CE family 1 esterases from anaerobic micro‐organisms such as Orpinomyces sp., Ruminococcus albus and Clostridium thermocellum. The gene comprised 828 nucleotides encoding a polypeptide of 275 amino acids. The coding sequence was cloned into the pET30a expression vector and overexpressed in E. coli BL21 (DE3). Gene product Fae1A was found to exhibit activity against a number of substrates including naphthyl fatty acid esters, p‐nitrophenyl fatty acid esters and hydroxylcinnamic acid esters. Conclusions: Fae1A exhibited a lower K_m and higher catalytic efficiency (k_cat/K_m) on ferulic acid esters than on α‐NA or p‐nitrophenyl acetate, suggesting that it has a higher affinity for ethyl and methyl ferulate than for the acetyl esters. It releases ferulic acid and p‐coumaric acid from barley straw. Activity of Fae1A was inhibited by the serine‐specific protease inhibitor, phenylmethylsulfonyl fluoride, indicating that a serine residue plays a role in its activity. Significance and Impact of the Study: To our knowledge, this is the first report of characterization of carbohydrate esterase gene from the genus of Anaeromyces.     

Characterization and partial purification of indole-3-butyric acid synthetase from maize (Zea mays)

In previous work it has been shown that the route from indoleacetic acid (IAA) to indolebutyric acid (IBA) is likely to be a two-step process with an unknown intermediate designated ‘product X′. Our objective was to characterize and purify enzyme activities that are involved in these reactions. Indole-3-butyric acid synthetase was isolated and characterized from light-grown maize seedlings (Zea mays L.), which were able to synthesize IBA from indole-3-acetic acid (IAA) with ATP and acetyl-CoA as cofactors. The enzyme activity is most likely located on the membranes of the endoplasmic reticulum, as shown by means of aqueous two-phase partitioning and sucrose density gradient centrifugation, with subsequent marker enzyme analysis. It was possible to solubilize the enzyme from the membranes with a detergent (CHAPS) and high concentrations of NaCl. The molecular mass of solubilized IBA synthetase was ca 31 kDa and its isoelectric point was at pH 4.8. The enzyme forming the reaction intermediate had a molecular mass of only 20 kDa and it seemed to be located on different membranes. Inhibition experiments with reducing agents and sulfhydryl reagents indicated that no sulfhydryl groups or disulfide bridges were present in the active centre of IBA synthetase. KCN inhibited the enzyme activity completely, and sodium azide by about 50%. Substrate analogs. such as 1-IAA, 2,4-dichlorophenoxyacetic acid, phenylacetic acid, and naphthaleneacetic acid, inhibited IBA formation to a high extent. Experiments with tunicamycin gave evidence that the enzyme is not a glycoprotein. These findings were confirmed by affinity chromatography with Concanavalin A. where the enzyme did not bind to the matrix. Further purification of the IBA synthetase on an ATP-affinity column resulted in a more than 1 000-fold purification compared to the microsomal membranes. IBA synthetase activity was also present in other plant families. Our results present further evidence that IBA is synthesized by a two-step mechanism involving two different enzyme activities.     

玉米种子萌发过程幼叶细胞中淀粉粒的积累观察

研究玉米萌发初期幼叶的发育。在幼叶不同的发育时期 ,分别用 PAS反应 ,考马氏蓝处理不同叶片 ,结果发现 :叶片细胞内的叶绿体在叶片即将抽出时才形成 ;从浸种萌动到叶片进行光合作用前 ,植株的营养供给 ,主要靠叶片自身淀粉粒的积聚提供 ;在幼叶抽出以前 ,胚芽鞘的薄壁细胞中布满淀粉粒 ,随着叶片的发育 ,这些淀粉粒逐渐减少 ;而幼叶中的淀粉粒的变化情况正好相反 :在种子萌发初期 ,幼叶细胞内只有少量的淀粉粒 ,以后淀粉粒的积累逐渐增多 ;在这个阶段无蛋白质的积聚。幼叶中维管束的发生是先中间后两边 ,维管束中的韧皮部先形成 ,木质部后发生。