A deep learning-integrated phenotyping pipeline for vascular bundle phenotypes and its application in evaluating sap flow in the maize stem

Plant vascular bundles are responsible for water and material transportation, and their quantitative and functional evaluation is desirable in plant research. At the single-plant level, the number, size, and distribution of vascular bundles vary widely, posing a challenge to automatically and accurately identifying and quantifying them. In this study, a deep learning-integrated phenotyping pipeline was developed to robustly and accurately detect vascular bundles in Computed Tomography(CT) images of stem internodes. Two semantic indicators were used to evaluate and identify a suitable feature extraction network for semantic segmentation models. The epidermis thickness of maize stem was evaluated for the first time and adjacent vascular bundles were improved using an adaptive watershed-based approach. The counting accuracy(R^(2)) of vascular bundles was 0.997 for all types of stem internodes, and the measured accuracy of size traits was over 0.98. Combining sap flow experiments, multiscale traits of vascular bundles were evaluated at the single-plant level, which provided an insight into the water use efficiency of the maize plant.

A Preliminary Study on a Specifically Expressed Arabidopsis Promoter in Vascular Bundle

From a population of about 3500 single plants in Arabidopsis promoter trapping bank, one plant whose GUS-gene had been specifically expressed in vascular bundle, was screened by the method of gus tissue staining. The T-DNA flanking sequence was amplified using TAIL-PCR. This band will be purified and connected to TA cloning vector. After sequencing and searching in the genebank, its function will be demonatrated through transformation.

Study on the Relationship of the Characteristics of Vascular Bundles in the First Internode and Grain-filling of Heavy Panicle Type Hybrid Rice

The characteristics of vascular bundle in the first internode from top and grain-filling of heavy panicle type hybrid rice(HPTHR) were studied. The results were as follows: The HPTHR had more vascular bundles and a much bigger area of single vascular bundle, all vascular bundle, all phloem and all xylem in the first inter-node than Shanyou63. The vascular bundles had the similar load of spikelet number and sink capacity between the HPTHR and Shanyou63. The HPTHR had not only a larger sink but also normal grain-filling and sink-filling, high seed-setting rate and heavier panicle. Those characteristics of vascular bundle were the biological bases for the larger and heavier panicle of HPTHR. The results also indicated that the breeding model of HPTHR was an effective measure for the super high-yielding rice breeding.

Vascular bundle connection between seed stalk and seed coat of Caragana arborescens

The purpose of this study was to investigate the morphology, transmission route of photosynthetic products in the seed stalk of Caragana arborescens Lam. at various stages after flowering and evaluate the role of the seed stalk in seed maturation using scanning electron microscopy. We found that the mature seed stalk mainly consisted of vessels,sclerenchymatous cells and parenchymatous cells. Elongated cells formed a ring at the junction between the seed stalk and seed, and a large hollow was produced by two layers of sclerenchymatous cells in the interior seed, which functions to sustain seed growth. The opening in the center of the ring was closed during an early stage of seed development, and the opening enlarged as the seed matured.There were two routes for material transport, which started from the seed stalk： one from the seed coat to the embryo,and another directly entering the embryo. Vessels, which are abundant in the seed stalk, are the pathway for absorption of nutrients by the seeds. The vessels were densely packed in the seed stalk, and secondary thickening of the cell wall of the vessels was characterized by net thread thickening, while the secondary thickening of the cell wall of the vessels within the seed was characterized by screw thread thickening. The morphological characteristics of the vessels were adaptive to its functions.

QTLs analysis of rice peduncle vascular bundle and panicle traits

The vascular bundle in plants plays an important role intransportation of photosynthetic products, mineral nutri-ents, water, arid so on. Significant positive correlationswere found between grain yield, panicle traits and the No.of peduncle vascular bundles. So, it is very important tostudy the inheritance of peduncle vascular bundle, whichis a quantitative trait.

Identification and fine mapping of quantitative trait loci for the number of vascular bundle in maize stem

Studies that investigated the genetic basis of source and sink related traits have been widely conducted.However, the vascular system that links source and sink received much less attention. When maize was domesticated from its wild ancestor, teosinte, the external morphology has changed dramatically; however, less is known for the internal anatomy changes. In this study, using a large maize-teosinte experimental population, we performed a high-resolution quantitative trait locus（QTL） mapping for the number of vascular bundle in the uppermost internode of maize stem.The results showed that vascular bundle number is dominated by a large number of small-effect QTLs, in which a total of 16 QTLs that jointly accounts for 52.2% of phenotypic variation were detected, with no single QTL explaining more than 6% of variation. Different from QTLs for typical domestication traits, QTLs for vascular bundle number might not be under directional selection following domestication.Using Near Isogenic Lines（NILs） developed from heterogeneous inbred family（HIF）, we further validated the effect of one QTL qVb9-2 on chromosome 9 and fine mapped the QTL to a 1.8-Mb physical region. This study provides important insights for the genetic architecture of vascular bundle number in maize stem and sets basis for cloning of qVb9-2.

Improved maceration techniques to study the fruit vascular anatomy of grape

The visualization and evaluation of fruit vascular bundles in grape are indispensable for understanding the development and growth of the fruit. The vascular bundles in grape are as thin as human hair, and strongly adhere to flesh cells, making it difficult to isolate individual intact vessel elements. Currently there is little information about methods to characterize grape vascular tissue. In this study, we describe an easy and effective technique to visualize the xylem cell structure of the ‘Red Globe' grape(Vitis L.). The intact vascular bundles of the grape were isolated through enzymatic degradation of flesh cells by hydrolases which were effective in removing flesh cells from vascular bundles. This enabled the illustration of the fine surface structure of vessel elements and their arrangement in the vascular bundles of the grape. This modified method to separate the vascular bundles into individual vessel elements was more effective than former methods of manually shaking to isolate individual vessels. Clear images of xylem vessel arrangement and structural characteristics of individual vessel element were acquired by light microscopy(BX51, Olympus, Tokyo, Japan), transmission electron microscopy(Tecnai 12, Philips, Netherlands) and scanning electron microscopy(GeminiSEM 300, Carl Zeiss, Germany). In addition, the 3D structure of vessel elements was observed using confocal laser scanning microscopy(Zeiss: model: LSM-880, Oberkochen, Germany). The imaging techniques for visualizing and analyzing the structure of xylem vessels in grape described in this study are an improvement of past methods. An effective method to isolate grape vascular bundles from flesh cells was also developed, which enables the imaging of the fine surface structure of vessel elements and their arrangement in grape vascular bundles. By adjusting the process of maceration of vascular bundles, an easy and effective method was developed to gently disrupt the plant tissue and isolate individual vessel elements. These improved techniques are suitable to observe the intact morphology of vascular bundles in the grape, improve experimental efficiency and accelerate new discoveries on the anatomical structure of the grape.

Irrigation regimes modulate non-structural carbohydrate remobilization and improve grain filling in rice(Oryza sativa L.)by regulating starch metabolism

Recently developed ‘super’ rice cultivars with greater yield potentials often suffer from the problem of poor grain filling, especially in inferior spikelets. Here, we studied the activities of enzymes related to starch metabolism in rice stems and grains, and the microstructures related to carbohydrate accumulation and transportation to investigate the effects of different water regimes on grain filling. Two ‘super’ rice cultivars were grown under two irrigation regimes of well-watered(WW) and alternate wetting and moderate soil drying(AWMD). Compared with the WW treatment,the activities of ADP glucose pyrophosphorylase(AGPase), starch synthase(StSase) and starch branching enzyme(SBE), and the accumulation of non-structural carbohydrates(NSCs) in the stems before heading were significantly improved, and more starch granules were stored in the stems in the AWMD treatment. After heading, the activities of α-amylase, β-amylase, sucrose phosphate synthase(SPS) and sucrose synthase in the synthetic direction(SSs)were increased in the stems to promote the remobilization of NSCs for grain filling under AWMD. During grain filling, the enzymatic activities of sucrose synthase in the cleavage direction(SSc), AGPase, StSase and SBE in the inferior spikelets were increased, which promoted grain filling, especially for the inferior spikelets under AWMD.However, there were no significant differences in vascular microstructures. The grain yield and grain weight could be improved by 13.1 and 7.5%, respectively, by optimizing of the irrigation regime. We concluded that the low activities of key enzymes in carbon metabolism is the key limitation for the poor grain filling, as opposed to the vascular microstructures, and AWMD can increase the amount of NSC accumulation in the stems before heading, improve the utilization rate of NSCs after heading, and increase the grain filling, especially in the inferior spikelets, by altering the activities of key enzymes in carbon metabolism.

Identification of Mucilage Cavity as a Significant Microscopic Characteristic Existing in Phloem Instead of Pith of Radix et Rhizoma Rhei

The morphologic and microscopic features of Radix et Rhizoma Rhei were studied. The study verified that the mucilage cavities did exist in phloem of rhizomes and roots or abnormal vascular bundles. Also, they were in similar distribution in three species of Radix et Rhizoma Rhei. The diagnostic characteristic for microscopic identification was found to be the similar distribution of abnormal vascular bundles in pith of rhizomes in all three species. And the appearance of the crude drug varied more depending on the plants＇ geographical origin and different preliminary treatment on the spot of collection than on the species differences. Our findings, having not been delineated clearly so far in the previous reports, are helpful for clarifying current descriptions in different literatures or standards and make a full understanding on microscopic and macroscopic identification of Radix et Rhizoma Rhei.

Comparison of Morphological and Genetic Differentiations in Filial Generation of Cross Between Indica and Japonica Rice

A recombinant inbred line （RIL） population of F8 and F9 generations derived from a cross between a typical indica rice （Qishanzhan） and a typical japonica rice （Akihikari） was used to study the difference between morphological differentiation based on phenotype characters and genetic differentiation using indica and japonica specific SSR markers, and to evaluate the relationship between vascular bundle characters and morphological and genetic differentiations. The results showed that the frequency distributions of morphological and genetic differentiations were all inclined to japonica type in the filial generation. The population was more inclined to japonica type based on genetic differentiation than on morphological differentiation. The consistent degrees of classification based on the Cheng’s index, the ratio of large vascular bundle number to small vascular bundle number in panicle neck （RLSVB） and the ratio of large vascular bundle number in the second internode from the top to that in the panicle neck （RLVB） were all about 50% compared with the genetic differentiation, and the consistent degree of the total scores of the Cheng’s index combined with the vascular bundle number ratios was significantly increased to about 80% compared with the genetic differentiation. Therefore, the vascular bundle characters could be used as a helpful supplement for subspecies classification.

Effects of Acetylcholine, Cytochalasin B and Amiprophosmethyl on Phloem Transport in Radish （Raphanus sativas）

We investigated the role of the ＂sieve tube-companion cell complex＂ lining the tube periphery, particularly the microfilament and microtubule, in assisting the pushing of phloem sap flow. We made a simple phloem transport system with a living radish plant, in which the conducting channel was exposed for local treatment with chemicals that are effective in modulating protoplasmic movement （acetylcholine, （ACh） a neurotransmitter in animals and insects; cytochalasin B, （CB） a specific inhibitor of many cellular responses that are mediated by microfilament systems and amiprophos-methyl, （APM） a specific inhibitor of many cellular responses that are mediated by microtubule systems）. Their effects on phloem transport were estimated by two experimental devices： （i） a comparison of changes in the amount of assimilates in terms of carbohydrates and ^14C-labeled photosynthetic production that is left in the leaf blade of treated plants; and （ii） distribution patterns of ^14C-labeled leaf assimilates in the phloem transport system. The results indicate that CB and APM markedly inhibited the transfer of photosynthetic product from leaf to root via the leaf vein, while ACh enhanced the transfer of photosynthetic product in low concentrations （5.0×10^-4 mol/L） but inhibited it in higher concentrations （2.0×10^-3 mol/L） from leaf to root via the leaf vein. Autoradiograph imaging clearly reveals that ACh treatment is more effective than the control, and both CB and APM treatments effectively inhibit the passage of radioactive assimilates. All of the results support the postulation that the peripheral protoplasm in the sieve tube serves not only as a passive semi-permeable membrane, but is also directly involved in phloem transport.

Effects of Anatomical Characteristics of Ethiopian Lowland Bamboo on Physical and Mechanical Properties

The main aim of this study was to evaluate the effects of anatomical characteristics of Ethiopian lowland bamboo on selected physical and mechanical properties. A total of 45 solid culms from three different age groups (2-, 3- and 4- year-old) were harvested from natural bamboo forest in Ethiopia and then samples were transported to China for carrying out anatomical characteristics test. Physical and mechanical properties testing were conducted in Ethiopia. The result indicates that age and height had significant effects on fiber length, fiber diameter and fiber wall thickness and insignificant effects on vascular bundles concentration. More over, fiber length, fiber diameter and vascular bundle size have a significant effect on both properties of this solid culm. The density and moisture content (MC) were highly affected by age and height variations. Density increased with an increase of age and MC decreased with an increase of age. The higher values of modulus of rupture (MOR) and modulus of elasticity (MOE) were obtained in the middle portions of height. Both values showed decreasing trend towards the top portion. Compression strength of culms decreases with increases of age and height. The results of correlation analyses revealed that the fiber length, the fiber diameter and the fiber wall thickness all had a positive correlation with the density and a negative correlation with MC. All anatomical variables showed insignificant correlation with all the shrinkages except the fiber wall thickness that showed a negative correlation with the radial shrinkage. The result further revealed a positive correlation of density with MOR and a negative correlation with MOE. The fiber length also showed a positive correlation with MOE. Both the fiber diameter and the vascular bundle size showed a positive correlation with the compression strength. However, MC showed a negative correlation.

Soda pulping of umbrella palm grass (cyperus flabettiformic)

Chemical,morphological and anatomical characteristics of Cyperus flabettiformic were evaluated.It is characterized with lowα-cellulose(32.2%)and moderate lignin(24.0%)content.Fiber length of C.flabettiformic was shorter(0.94mm)but slender ratio was extremely high(166).Anatomically,this grass is constituted by homogeneous parenchyma cells and the vascular bundles(11.8%).Delignification of C.flabettiformic was carried out by conventional soda pulping under varying alkali charge,time and liquor to material ratio at the boiling temperature.Total pulp yield was 55.0%with kappa number 15.5 at the cooking conditions of 12%alkali charge,2 h of cooking and liquor to material ratio 10:1.Multivariate data analysis was used to evaluate the influence of alkali charge,cooking time and liquor ratio on pulp yield and kappa number.Alkali charge had a significant effect on pulp yield and kappa number.PLSR model showed better prediction efficiencies for pulp yield and PCR model performed better prediction for kappa number.Initial drainage resistance and papermaking properties of C.flabettiformic pulp were higher than other nonwood.