In vitro screening of mulberry (<Emphasis Type="Italic">Morus</Emphasis> spp.) for salinity tolerance

An efficient in vitro screening method has been developed for mulberry (Morus spp. ) to screen salinity-tolerant genotypes from a large population. Axillary buds from field-grown plants were cultured on MS medium containing five different concentrations (0.0%, 0.25%, 0.5%, 0.75% and 1.00%) of sodium chloride (NaCl) in order to study the shoot growth pattern. Rooting was also tested at four different concentrations of NaCl (0.0%, 0.1%, 0.2% and 0.3.%). NaCl has been found to inhibit the growth and development of mulberry shoots and roots in vitro. The survivability of the axillary buds of the genotypes tested was reduced from 83.7% for the controls to 6.1% in 1.0% NaCl. The average number of roots developed by the genotypes ranged from 11.9 (controls) to 0.2 (0.3% NaCl). Out of the 63 genotypes tested, only seven—Rotundiloba, English black, Kolitha-3, Berhampore-A, Kajli, BC₂59 and C776—developed roots in 0.3% NaCl. Root growth was also reduced drastically from 1.8 cm for the controls to 0.1 cm in 0.3% NaCl. To test the reproducibility of the results in soil, five tolerant and two susceptible genotypes, identified in this in vitro study, were selected and tested under ex vitro conditions. The significant correlation coefficients obtained between the performances of these genotypes under both types of cultural conditions revealed that in vitro screening of mulberry through axillary bud culture is an easy and efficient method to identify salt-adapted genotypes within a limited space and time period.Abbreviations BA 6-Benzylaminopurine - dS m^-1 Deci-Siemens per meter - EC Electric conductivity - NAA -Naphthaleneacetic acidCommunicated by G.C. Phillips     

Comparative proteomic analysis provides new insights into mulberry dwarf responses in mulberry (Morus alba L.)

Mulberry dwarf (MD) is a serious infectious disease of mulberry caused by phytoplasma. Infection with MD phytoplasma results in stress phenotypes of yellowing, phyllody, stunting, proliferation, and witches' broom. Physiological and biochemical analysis has shown that infection with MD phytoplasma causes an increase in soluble carbohydrate and starch content, and a decrease in the net photosynthesis rate, carboxylation efficiency, and pigment content of leaves. Furthermore, damage to the chloroplast ultrastructure was detected in infected leaves. To better understand the pathogen‐stress response of mulberry (Morus alba L.) to MD phytoplasma, we conducted a comparative proteomic analysis using 2‐DE of infected and healthy leaves. Among 500 protein spots that were reproducibly detected, 20 were down‐regulated and 17 were up‐regulated. MS identified 16 differentially expressed proteins. The photosynthetic proteins rubisco large subunit, rubisco activase, and sedoheptulose‐1,7‐bisphosphatase showed enhanced degradation in infected leaves. Based these results, a model for the occurrence mechanism of MD is proposed. In conclusion, this study provides new insights into the mulberry response to MD phytoplasma infection.     

In vivo growth of encapsulated axillary buds of mulberry (Morus indica L.)

Axillary buds excised from aseptic shoot cultures of mulberry were encapsulated in an alginate matrix under non-aseptic conditions. Addition of a fungicide to the alginate beads prevents contamination of the bud and increased survival of the buds when sown in soil.     

Chemical characterization of cytotoxic indole acetic acid derivative from mulberry fruit (Morus alba L.) against human cervical cancer

The fruit of the white mulberry tree (Morus alba L.) is a multiple fruit with a sweet flavor commonly consumed around the world. Chemical investigation of the fruits led to the isolation of two indole acetic acid derivatives (1 −2) including a new compound, which turned out to be an isolation artifact, 3S-(β-D-glucopyranosyloxy)-2,3-dihydro-2-oxo-1H-indole-3-acetic acid butyl ester (1), along with five known compounds (3 −7). Compounds 2 and 7 were newly identified from mulberry fruit. The new isolation artifact (1) exhibited cytotoxic effect on human cervical cancer Hela cells in a dose-dependent manner. Compound 1 activated caspase-8, caspase-9, and caspase-3, followed by cleavage of PARP, a substrate of caspase-3, in a dose-dependent manner. Simultaneous alterations in protein expression of mitochondrial factors Bax, BID and Bcl-2 were also observed. A comparison between compounds 1 and 2 led to a structure-activity relationship analysis of the cytotoxic effect. These results suggest that compound 1 could be beneficial in human cervical cancer treatment, and provide a theoretical basis for further application of compound 1.     

Production of triploid plants of mulberry (Morus alba L) by endosperm culture

 A continuously growing callus was obtained from immature endosperm of Morus alba L Cv S-36 cultured on Murashige and Skoog medium containing 5 μm 2,4-dichlorophenoxyacetic acid. Shoot buds were produced when the callus was subcultured on a medium containing a cytokinin or a cytokinin and 1-naphthaleneacetic acid (NAA). The maximum number of shoots was formed on the medium containing thidiazuron (1 μM), or benzylaminopurine (5 μM) and NAA (1 μM). Shoots were multiplied by forced axillary branching and rooted in vitro. Endosperm-derived plants were established in soil. Each of the ten plants examined cytologically was triploid (3 n=42). Received:17 February 1999 / Revision received: 4 May 1999 / Accepted: 19 May 1999     

Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves

To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD₆₀₀ of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry.     

In vitro screening of some plant extracts against fungal pathogens of mulberry ( Morus spp.)

Twenty one plant species were screened in vitro for their fungitoxic properties against four fungal pathogens viz., Phyllactinia corylea (Powdery mildew), Peridiopsora mori (Brown rust) and Pseudocercospora mori (Black leaf spot) by slide germination method and Myrothecium roridum (Brown leaf spot) by poisoned food technique. Conidial germination of P. corylea was significantly reduced in 5% (w/v) ethanolic extracts all tested plant. Extract of Cassia tora and Cassia sophera completely inhibited conidial germination of P. corylea. Other effective plant extracts inhibited >?90% germination were Allium sativum (99.56%), Ocimum sanctum (97.80%), Moringa oleifera (97.32%). Conidial germination of Pseudocercospora mori was completely inhibited in extract of A. sativum and D. metel. More than 90% inhibition was observed with extract of Holarrhena antidysentrica (93.10%), Adhatoda Zeylanica (91.40%) and Calotropis gigentia (90.40%). Urediniospore germination of Peridiopsora mori was significantly reduced in 19 plant extracts. Extract of A. sativum and D. metel completely inhibited urediniospore germination P. mori. Other effective plant extracts, which inhibited >?90% germination were Calotropis gigentia (99.40%), Targets patula (98.96%), Azadirachta indica (98.85%), Mirabilis jalapa (95.50%) and Chromoleana odorata (90.51%). Maximum inhibition (33.33%) of colony growth of M. roridum was observed with amendment of 5% solvent extracts of D. metel followed by A. sativum (25%), Chromoleana odorata (20%) and Eucalyptus citriodora (16.66%).     

Apical dominance in mulberry (Morus alba): Effects of position of lateral and accessory buds and leaves

Suzuki, T. 1990. Apical dominance in mulberry ( Morus alba ): Effects of position of lateral and accessory buds and leaves. – Physiol. Plant. 78: 468-474.
Removing apical portions of current growth coppice shoots from field-grown, low-pruned stumps of mulberry ( Morus alba L. cv. Shin-ichinose) caused sprouting of one or more upper main buds, almost concurrently with that of accessory buds. However, removal of the new sprouts, including those from accessory buds, slightly enhanced the sprouting of buds immediately below them, and did not affect buds lower down. In contrast, mature leaves inhibited the buds in their axils. Budless, leafy nodes on the upper part of pruned shoots tended to swell after treatment, perhaps due to the accumulation of substances translocated from the roots and possibly from the remaining leaves. Lateral buds at different positions along the shoot differed in their sprouting ability with buds lower on the shoot being more inhibited. This inhibition gradient dissappeared when all coppice shoots on one stump were pruned to the same bud position, suggesting inhibition from neighboring, actively growing shoots. These results demonstrate that acropetal influences are important in bud dominance relationships.     

硝酸银对桑树遗传转化的作用(简报)

调查了硝酸银对农杆菌介导的桑（Morus alba L.)品种“新一之濑”遗传转化的作用。在抗性筛选培养基中添加2mg L^-1硝酸银,桑叶盘褐化死亡率减少7．2％,抗性芽分化率增加3．9％,外源基因转化频率增加9．6％,“假阳性”转化体减少42．5％。感染农杆菌的桑叶盘经MS培养基培养、农杆菌液体培养和固体培养基上划线培养等实验证实,硝酸银对工程农杆菌LBA4404的生长具有抑制作用,这可能是提高桑树转化频率的原因之一。     

Stress responses in two genotypes of mulberry (Morus alba L.) under NaCl salinity

Changes in biomass yield rates, cell membrane stability (CMS), malondialdehyde (MDA) content and in the levels of physiological stress markers such as proline and glycine betaine in two high yielding genotypes (S1 and ATP, salt tolerant and salt sensitive, respectively) of mulberry under NaCl salinity were studied. Biomass yield rates and CMS were significantly decreased in both the genotypes under stress conditions. Per cent of decrease in biomass yield rate and CMS was relatively less in S1 than in ATP. Salt stress results a significant increase in the accumulation of proline, by 6-fold in S1 and 4-fold in ATP. Glycine betaine content was also increased significantly in stressed plants. However, the per cent increase was more in S1 than in ATP. The level of lipid peroxidation as indicated by MDA formation was greater in ATP than in S1. These results clearly support the better salt tolerant nature of S1 compared to ATP genotype.     

Apical control of lateral bud development and shoot growth in mulberry (Morus alba)

Measurements of changes in the degree of dominance by upper laterals over lower ones in coppice shoots (1-year-old stems) of 12-year old low- pruned stumps of mulberry ( Morus alba L. cv. Shin-ichinose) were made by removal of upper stem sections (pruning) or of lateral buds (debudding.) before spring bud burst, as part of a study of the factors involved in dominance relationships between the developing buds and elongating shoots. Besides inhibition of lower laterals by the upper, leading shoots, there was evidence for mutual inhibition (competition) of neighboring laterals along the stem. Thus in stems in which every other bud, or 4 out of every 5 buds were removed, there was a delay in growth cessation of lower laterals and their greater elongation than in controls. Such competition was seen to exist even between the uppermost and sub-terminal laterals, since the former elongated more in the absence of the latter.
In contrast to high and middle pruned stems, the delay in sprouting of the buds in low-pruned stems resulted in limited elongation of the shoots from such buds. This inhibition was removed when all the stems on a stump were pruned to the same length, suggesting that it was associated with intact stems with actively growing laterals. Patterns of regrowth of the short shoots (lower laterals) after summer pruning (middle-pruned) depended on the extent of removal of other stems with vigorously growing, upper laterals. These results demonstrate that both acropetal and basipetal influences are important in bud and shoot dominance relationships.     

Changes in leaf organisation, photosynthetic performance and wood formation during ex vitro acclimatisation of black mulberry (Morus nigra L.)

Changes in anatomical organisation of the leaf, photosynthetic performance and wood formation were examined to evaluate the temporal and spatial patterns of acclimatisation of micropropagated slow-growing black mulberry ( Morus nigra L.) plantlets to the ex vitro environment. Leaf structure differentiation, the rates of net photosynthesis (P_n), transpiration (E) and stomatal conductance (g_s), and secondary xylem growth were determined in the course of a 56-day acclimatisation. Differentiation of palisade parenchyma was observed 7 days after transfer. At this stage, the rates of P_n, E and g_s reached maximum values, after which the rates of all three gas exchange parameters gradually decreased. The highest proportion of woody area occupied by vessels was also observed 7 days after transfer. An important feature of developing woody tissue is the difference in patterns of vessel distribution from the characteristic differentiation patterns of earlywood and latewood vessels in mature wood of ring-porous trees. Vessels with lumen areas over 3000 μm² were only differentiated in acclimatised plantlets, whereas vessels in stems sampled on days 0 and 7 had very small lumen areas of up to 560 μm². Full acclimatisation, observed 56 days after transfer to the ex vitro environment, was associated with the rapid growth of new in vivo formed leaves, very low rates of E and g_s, and much increased secondary xylem tissue within the stem area.     

High-speed pollen release in the white mulberry tree, Morus alba L

Anemophilous plants described as catapulting pollen explosively into the air have rarely attracted detailed examination. We investigated floral anthesis in a male mulberry tree with high-speed video and a force probe. The stamen was inflexed within the floral bud. Exposure to dry air initially resulted in a gradual movement of the stamen. This caused fine threads to tear at the stomium, ensuring dehiscence of the anther, and subsequently enabled the anther to slip off a restraining pistillode. The sudden release of stored elastic energy in the spring-like filament drove the stamen to straighten in less than 25 μs, and reflex the petals to velocities in excess of half the speed of sound. This is the fastest motion yet observed in biology, and approaches the theoretical physical limits for movements in plants. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. An erratum to this aticle is available at .     

Ultrastructural and immunochemical features of the cell wall sac formed in mulberry (Morus alba) idioblasts

A peculiar inward growth, named a “cell wall sac”, formed in mulberry (Morus alba) idioblasts, is a subcellular site for production of calcium carbonate crystals. On the basis of ultrastructural observations, a fully expanded cell wall sac could be divided into two parts—an amorphous complex consisting of multi-layered compartments with multiple fibers originating from the innermost cell wall layer, and a peripheral plain matrix with fiber aggregates. Immunofluorescent localization showed that low and highly esterified pectin epitopes were detected at the early stages of development of the cell wall sac, followed by complete disappearance from the both parts of fully enlarged mature sac. In contrast, the xyloglucan epitope remained in the compartment complex; this was supported by the observation that the xyloglucan epitope labeled with immuno-gold particles is found on fibers in the complex part.     

Accumulation of Flavonoid Glycosides and UFGT Gene Expression in Mulberry Leaves (Morus alba L.) before and after Frost

In order to determine the molecular mechanism underlying the influence of frost on chemical changes in mulberry leaves, the UFGT activity, expression level, and accumulation of flavonoid glycosides in mulberry leaves (Morus alba L.) were studied. The expression of UFGT gene was investigated by quantitative real‐time PCR (qRT‐PCR) and the UFGT activity, accumulation of flavonoid glycosides was studied by high performance liquid chromatography. Then, the correlation between the expression level of UFGT, the UFGT activity, and the flavonoid glycosides accumulation with temperature was explored. The accumulation of isoquercitrin and astragalin is significantly positively correlated with UFGT gene expression and UFGT activity. On the contrary, the average temperature was significantly negatively correlated with the level of UFGT gene expression and UFGT activity. The results show that after frost, low temperature can induce the expression of UFGT gene in mulberry leaves, resulting in the accumulation of flavonoid glycosides.     

Mulberry leaves (Morus alba L.) ameliorate obesity-induced hepatic lipogenesis,fibrosis, and oxidative stress in high-fat diet-fed mice

Obesity is associated with chronic diseases such as fatty liver, type 2 diabetes, cardiovascular disease, and severe metabolic syndrome. Obesity causes metabolic impairment including excessive lipid accumulation and fibrosis in the hepatic tissue as well as the increase in oxidative stress. In order to investigate the effect of mulberry leaf (Morus alba L.) extract (MLE) on obesity-induced oxidative stress, lipogenesis, and fibrosis in liver, MLE has been gavaged for 12 weeks in high-fat diet (HFD)-induced obese mice. MLE treatment significantly ameliorated LXRα-mediated lipogenesis and hepatic fibrosis markers such as α-smooth muscle actin, while MLE up-regulated lipolysis-associated markers such as lipoprotein lipase in the HFD-fed mice. Moreover, MLE normalized the activities of antioxidant enzymes including heme oxygenase-1 and glutathione peroxidase in accordance with protein levels of 4-hydroxynonenal in the HFD-fed mice. MLE has beneficial effects on obesity-related fatty liver disease by regulation of hepatic lipid metabolism, fibrosis, and antioxidant defense system. MLE supplementation might be a potential therapeutic approach for obesity-related disease including non-alcoholic fatty liver disease.     

Isolation of an 1-aminocyclopropane-1-carboxylate oxidase gene from mulberry (Morus alba L.) and analysis of the function of this gene in plant development and stresses response

Mulberry (Morus alba) is an important crop tree involved in sericulture and pharmaceuticals. To further understand the development and the environmental adaptability mechanism of mulberry, a cDNA of the gene MaACO1 encoding 1-aminocyclopropane-1-carboxylate oxidase was isolated from mulberry. This was used to investigate stress-responsive expression in mulberry. Developmental expression of ACC oxidase in mulberry leaves and spatial expression in mulberry flowers were also investigated. Damage and low-temperature treatment promoted the expression of MaACO1 in mulberry. In leaves, expression of the MaACO1 gene increased in cotyledons and the lowest leaves with leaf development, but showed reduced levels in emerging leaves. In flowers, the pollinated stigma showed the highest expression level, followed by the unpollinated stigma, ovary, and immature flowers. These results suggest that high MaACO1 expression may be predominantly associated with tissue aging or senescence in mulberry.     

Radiation damage and induced tetraploidy in mulberry (Morus Alba L.)

Vigorously growing mulberry shoots were exposed to 5 kR of gamma rays at the rate of 0·2 kR/hr and 5·0 kR/hr and successively pruned three times in two growing seasons. The most radiosensitive part of both the apical and axillary meristems was the second cell layer. The younger axillary bud primordia were more sensitive to radiation than the older ones. Recovery from radiation damage was assumed to be from the flank meristem in the shoot apex. The frequency of mutations was much lower than that of tetraploidy. Among the tetraploids 50% were 2-4-4 chimeras.     

In vitro fertilization in nonhuman primates

Ovulation induction, sperm capacitation, and fertilization have been studied for over 50 years in nonhuman primates but it has only been in the past 20 years that extensive studies on sizeable numbers of embryos have been carried out. Of over 200 species of nonhuman primates only a few have been studied and the majority of the findings come from studies of the squirrel monkey, baboon, rhesus, and cynomolgus macaque. Nevertheless, the fertilization process appears to be similar to that identified in other mammals and in man.