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     

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.     

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.     

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.     

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.     

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.     

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 .     

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.     

Genetic variability and association of ISSR markers with some biochemical traits in mulberry (Morus spp.) genetic resources available in India

Utilizing intersimple sequence repeat (ISSR) markers, 18 mulberry (Morus spp.) germplasm collections were studied for genetic variability, phylogenetic relationship, and association with protein and sugar content. The genetic polymorphism exhibited by ISSR primers was 100%, and the genetic diversity recorded among the mulberry accessions had an average of 0.263 ± 0.094. Dendrogram (unweighted pair group method analysis) clustered the mulberry accessions into two major groups, one comprised the accessions collected from north or northeast regions of India, and the other comprised three subclusters and one isolate, i.e., Assamjati, a collection from Assam. Another subcluster contained accessions collected from Kerala, which belong to Morus indica. These accessions of M. indica from Kerala were found to be genetically diverse from north and northeast India. Multidimensional scaling of the ISSR data clearly separated the mulberry accessions according to their genetic diversity and protein content. Mulberry accessions were arbitrarily grouped into three classes viz. very low, moderate, and high in terms of protein and sugar content using standard statistical programs. Stepwise multiple regression analysis identified four ISSR markers (835_1,600, 835_5,600, 822_2,500, and 807_2,500) associated with protein content with highly positive correlation (p < 0.001) with linear curves with high F values (18.055 to 48.674; p < 0.001). In case of sugar content, four ISSR markers viz. 812₉₀₀, 817_1,500, 826_1,500, and 810_8,000 showed negative correlation. Hence, DNA markers for proteins seem promising and may be used in marker-assisted breeding program.     

Phytostilbenoid production in white mulberry (Morus alba L.) cell culture using bioreactors and simple deglycosylation by endogenous enzymatic hydrolysis

Phytostilbenes are responsible for several biological activities of mulberry (Morus sp.), which has been widely used as a raw material in health products. This study aimed to investigate the capability of Morus alba L. cell in bioreactors to produce the major bioactive stilbenes. The cell obtained from air-driven bioreactors such as round bottom, flat bottom, and air-lift vessel shape bioreactors was collected and analyzed for the levels of mulberroside A and oxyresveratrol. The results showed that the cell culture in round bottom and air-lift vessel bioreactors had higher growth rate, as compared with the cell culture in shake flasks (1.38- and 1.41-fold, respectively). The optimized culture condition to produce mulberroside A was obtained from round bottom bioreactor culture (55.56 ± 11.41 μmol/L). Additionally, endogenous stilbenoid hydrolysis of cell from the bioreactor culture was examined. Under optimized hydrolytic conditions, mulberroside A in the cell was readily deglycosylated to give oxyresveratrol within 1 h. These results indicated that the glycoside mulberroside A in the cell is sensitive to the endogenous enzymatic hydrolysis. Interaction of the stilbenoid components with the endogenous hydrolytic enzyme triggered by cell disruption in M. alba samples was suggested to be the major cause of the alteration of the stilbenoid levels. These findings have provided a new approach to producing glycosidic compounds and corresponding aglycones in cell culture.

     

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 induction of tetraploid plants from diploid Zizyphus jujuba Mill. cv. Zhanhua

Tetraploid plants of Zizyphus jujuba Mill. cv. Zhanhua were obtained with in vitro colchicine treatment. Shoot tips from in vitro-grown plants were treated with five different concentrations of colchicine (0.01, 0.03, 0.05, 0.1, 0.3%) in liquid MS medium (Murashige and Skoog 1962), and shaken (100 rpm) at 25 °C in darkness for 24, 48, 72 or 96 h, respectively. Tetraploids were obtained at a frequency of over 3% by using 0.05% colchicine (48 h, 72 h) and 0.1% colchicine (24 h, 48 h) treatment as determined by flow cytometry. Cytological and morphological evidence confirmed the results of flow cytometric analysis. The chromosome number of diploid plants was 24 and that of tetraploid plants was 48. The stomata sizes of tetraploid plants were significantly larger than those of diploid plants, while the frequency of stomata were reduced significantly. Similarly, the chloroplast number of guard cells of tetraploid plants increased significantly. The selected tetraploid plants were grafted onto mature trees of Z. jujuba Mill. cv. Zhanhua in the field, resulted in thicker stems, rounder and succulent leaves, larger flowers and a delay in florescence time (3–4 days later) than diploid plants.     

In vitro hermaphrodism induction in date palm female flower

This study explores and reports on the gain brought to the morphogenetic aptitude of female date palm inflorescences through in vitro hermaphrodism induction. It investigates the main factors involved in the process of sex modification through hormonal induction, such as the floral developmental stage and hormone combination and concentration. It demonstrates that the vestigial stamens (staminodes) of female date palm flowers display a new and high capacity to proliferate under particular in vitro conditions, without blocking carpel's development, leading to morphologically typical hermaphrodite flowers. This de novo activation of repressed stamens was found to occur rapidly. The isolated pollen mother cells appear in the obtained anther's locules and undergo an ordinary microsporogenesis process. The data show that hermaphrodism induction depended heavily on both the growth regulators applied and the flower's developmental stage. They also confirm the early theory that suggests that dioecious plants come from a hermaphrodite ancestor. Such hermaphrodism control can provide new prospects and opportunities for the investigation of the in vitro self-fertilization process. It can also be useful in improving the understanding of the genetic mechanism involved in sex organ development in date palm.     

In vitro response of promising mulberry (Morus sp.) genotypes for tolerance to salt and osmotic stresses

An in vitro technique for screening mulberry genotypes tolerant to salt and osmotic stress has been standardized.Five mulberry genotypes, namely G2, G3, G4 along with control varieties i.e., S34 and S13, were tested on salt and osmotic stress media. Out of 14 media combinations tested, the optimum responses were observed on Kn 1 mg/l, in the case of G3 genotype, on Kn 2 mg/l with G2 genotype, on BAP 1 mg/l with G4 genotype and on BAP 2 mg/l with S34 and S13 genotypes.With regard to their performance on salt-stress media fortified with NaCl (0.1--2.0%), Na₂CO₃ + NaHCO₃ (pH 8.5--10.0), the data revealed that the genotype G4 ranked the highest in terms of sprouting percentage and shoot length, in comparison to the control variety (S34) at each concentration of NaCl up to 1.0% and pH up to 9.5. However, in the case of osmotic stress condition (media supplemented with 1.0--10.0% PEG),the control variety i.e. S13 itself exhibited the highest sprouting percentage and shoot growth compared to the other test genotypes. The genotype G4 has been screened as a salt tolerant genotype which can be tested under respective in vivo condition.