Abstract In this study， the plant biomass production， biomass translocation rates across tissues and the lodging resistant??associated traits of millet （Setaria italica L.） in North China were investigated. Among the four summer millet cultivars， Baogu 19 exhibited improved plant biomass （PB） production at flowering and maturity stages， biomass translocation amount （BTA） from vegetative tissues to seeds during filling period， and lodging resistant??associated （LRA） traits compared with other cultivars， including enhanced stem lignin contents， increased anti??broken resistance （ABR）， anti??puncturing resistance （APR）， and stem diameter （SD） of plants. Compared with treatment regular cultivation （RC）， high fertility treatment （HF） increased the plant BP， BTA from vegetative tissue to seed at filling stage， and the plant LRA traits； whereas high density treatment （HD） decreased the plant BP at plant level， plant BTA from vegetative tissues to seeds at filling stage， and the plant LRA traits. Correlation analysis revealed that stem ABR is significantly correlated with the plant lodging resistant??associated traits including APR and SD in the summer millet cultivars examined under various cultivation treatments. Our investigation indicates that cultivar Baogu 19 together with suitable fertilization and density can promote the plant biomass production， enhance vegetative tissue biomass translocation to seeds， and improve the lodging resistance of summer millet plants in North China.
　　Key words Millet （Setaria italica L.）， cultivation condition， plant biomass， biomass translocation rate， anti??lodging resistance
　　Millet （Setaria italica L.） is an old crop species cultivated widely around the world， which is suitable to be planted in regions with low soil fertility as well as inadequate water supply conditions[1]. Foxtail millet， the earliest domesticated type of millet， acts as the one being largely planted in China and East Asia countries. Millet production contributes greatly to human feeds in North China and the temperate zones of Asia and Europe[2-5] given that this crop species shares elite adaptation to diverse abiotic stresses[6].
　　The millet biomass production and yield are affected by the genetic and environmental factors. Genetic background of cultivars and external factors in growth conditions， such as light intensity， rainfall amount or irrigation， and inorganic nutrient status during plant growth and development impact on the productivity through modulating conversion efficiency of light energy， plant biomass production and biomass distribution among the tissues[7]. 　　Lodging resistance of plants at late growth stage is potential in impacting on the productivity of cereal crops. Plant lodging during filling stage in millet and other cereals due to permanent tilting or bending of stems is frequently caused by abnormal weather， such as rainstorm， leading to reduced biomass production and lowered yield together with difficult harvest[8-10]. Moreover， lodging occurred in cereals at the filling stage has resulted in potential health risk because grains infected by fungi can cause the mycotoxin accumulation in kernels[9，11-12]. Alleviation of plant lodging thus can effectively promote the production potential of millet as well as other cereal crop species.
　　Previously， the plant biomass production， biomass translocation property of vegetative tissues to harvest organ during filling stage， and the lodging resistant traits have been investigated in millet species[13-15]. However， most of them were performed focusing on the spring millet. The plant biomass production and biomass translocation characterization， especially the lodging resistant??associated traits in the summer millet， namely， a cultivation system in which millet is sown at summer followed by winter wheat and harvested at autumn in North China， remain largely to be further elucidated. In this study， using Baogu 19， its parents Jigu 19 and 9050 together with a cultivar planted currently in Hebei Plain named 60D as materials， we investigated the plant biomass production， biomass translocation property from vegetative tissues to harvest organ， and the lodging resistant??associated traits under different cultivation techniques， including regular cultivation （RC）， high fertilization （HF）， and high density （HD）. Extensive effects on above traits were observed under various cultivation techniques and Baogu 19 exhibited much more plant biomass， higher biomass translocation amount， and more improved lodging resistance??associated traits than other cultivars， especially under high density condition， suggesting its cultivation potential in North China as well as other similar ecological zones.
　　Materials and Methods
　　Plant materials and experiment arrangement
　　Totally four summer millet cultivars， including Baogu 19， Jigu 19， 9050， and 60D were used in this study. Among them， Baogu 19 is derived from Jigu 19 （female parent） and 9050 （male parent）. 60D is a control cultivar cultivated currently in Hebei plain， China. Field experiments were performed during 2015 and 2016 growth seasons at experimental station of Baoding Academy of Agricultural Sciences， Baoding， China （location of East longitude of 115.47 and North latitude of 38.47）. Soil type used for experiment is clay loam and the soil contains organic matter of 1.52%， total nitrogen of 0.082%， available nitrogen of 64.30 mg/kg， available phosphate 13.86 mg/kg， and exchangeable potassium of 112.53 mg/kg. The plots were arranged in a split plot design， using cultivation techniques （regular cultivation， RC； high fertility， HF； high density， HD） as main plot whereas cultivars （Baogu 19， Jigu 19， 9050， and 60D） as sub??main plot. As to the cultivation treatments， RC was supplied with basal N 75.00 kg/hm2， P2O5 109.31 kg/hm2， and K2O 90.38 kg/hm2； HF was established by using more basal inorganic nutrients， including basal N 150.00 kg/hm2， P2O5 218.62 kg/hm2， and K2O 180.76 kg/hm2， whereas HD established by increased plant density together with same amount of base fertilizer as RC. The plant density for RC and HF was 600 000/hm2 and for HD was 900 000/hm2. The experimental field was ploughed conventionally after winter wheat harvest on June 13， 2015 and June 12， 2016， respectively， and then fertilized as planed nutrient amounts. Seeds of the cultivars examined were sown on June 19， 2015 and June 18， 2016， respectively， with a 40 cm row distance. Plant densities in each plot were manually established at four??leaf stage according to the designed numbers. Other cultivation techniques during growth seasons， such as removal of weeds and chemical control for diseases and pests， were conducted following the conventional methods. 　　Assays of biomass and biomass translocation rates of vegetative tissues at flowering and maturity stages
　　At both flowering stage and maturity stage， twenty plants were collected from each plot to be subjected to evaluation of plant biomass and biomass translocation rate （BTR） of the vegetative tissues. For that， the leaves， stems， leaf sheaths， and spikes in plant samples were separately departed and oven dried at 80 ?? for 48 h. Tissue mass was obtained by weighing the dried tissue using electronic balance. Biomass at both stages was obtained by summing up the tissue masses at plant level and by multiplying plant biomass with density at plant area level. Biomass reduction amount （BRA） at late stage in each tissue at plant level or planting area level was calculated by minus the biomass at flowering stage to that at maturity stage. Biomass translocation rate （BTR） of vegetative tissues （i.e.， leaf， stem， and leaf sheath） were calculated based on follow formula：
　　BPR = （Biomass at flowering stage-Biomass at maturity stage）/（Spike weight at maturity stage??100）.
　　Assay of plant lodging resistant??associated traits
　　At mid??filling stage （20 d after flowering）， fifteen plants were collected from each plot to be subjected to characterization of the lodging resistant??associated traits of plants. The traits assessed included plant height （PH）， plant balancing position （BP） （balancing point of plant based on biomass， ratio of BP to PH， stem length （SL）， diameter （SD）， anti??broken resistance （ABR）， anti??puncturing resistance （APR）， and lignin content. Among these， PH， BP， ratio of BP to PH， SL， and SD were evaluated as described by Li et al.[16]； stem ABR and APR were assessed as described by Hu et al.[17]； and stem lignin contents were evaluated based on a histochemical staining approach as described by Okuno et al.[18].
　　Assay of the yield formation traits and yields
　　At maturity， thirty representative plants were collected from each plot and subjected to assay of the yield formation traits and yields. To that， the plant samples were firstly dried in room and spike weights obtained using electronic balance. Per 1 000??kernel weights were obtained by weighing representative one thousand kernels derived from typical spikes. The yields from the cultivars under various treatments were obtained by harvesting per planting area seeds at maturity.
　　Statistical analysis
　　The averages， standard errors， and statistical calculations as well as the correlation analysis were performed using the SAS software （SAS 12.1）. Due to similar results on assessed traits obtained in two growth seasons， data from the 2016 growth season were used in this investigation. 　　Results
　　Plant biomass at flowering and maturity stages
　　The plant biomass at flowering and maturity stages in cultivars grown under cultivation treatments of regular cultivation （RC）， high fertility （HF）， and high density （HD） are shown in Table 1. Among the treatments， much more biomass per plant and per planting area are shown in HF， suggesting that suitably increasing basal fertilizer can effectively improve plant biomass production at late growth stages. As to HD， the plant biomass were lower than RC at per plant level and higher than RC at per planting area， suggesting suitably increased density can promote plant biomass production at the plant area level. Among the cultivars， Baogu 19 showed the highest plant biomass at both flowering and maturity stages， followed by Jigu 19 and 60D， and the lowest in 9050 （Table 1）. Therefore， it can be concluded that Baogu 19 together with suitable fertilization and density can effectively improve the plant biomass production at late growth stages.
　　Biomass of vegetative tissues during filling stage
　　The biomass in various vegetative tissues at flowering stage and maturity stage， including tissues leaf， stem， and leaf sheath， are shown in Table 2. In consistent with plant biomass as aforementioned， each vegetative tissue possessed the highest biomass at these two stages under HF at both plant level and planting area level. Likewise， RC had higher tissue biomass at plant level and lower tissue biomass at plant area level than HD. Among the cultivars， Baogu 19 also behaved the highest vegetative tissue biomass at both flowering and maturity stages， followed by Jigu 19 and 60D， and 9050 （Table 2）. These results indicate that the vegetative tissue biomass in cultivars examined under different cultivation conditions corporately impact on the plant biomass.
　　Biomass translocation rates of the vegetative tissues during filling stage
　　Biomass translocation??associated traits in vegetative tissues during filling stage were calculated based on vegetative tissue biomass at flowering stage and those at maturity stage. The biomass reduction amount （BRA） and biomass translocation rate （BTR） in leaf， stem， and leaf sheath are shown in Table 3. Among the cultivation treatments， HF showed the highest BRA in vegetative tissues （i.e.， leaf， stem， and sheath） across cultivars， followed in RC， and the lowest in HD， suggesting that increased fertilizer amount enhances and plant density lowers the BRA of vegetative tissues during filling stage. Among the cultivars， no significant variation on BTR of vegetative tissues was observed， albeit that the cultivars with improved plant biomass at both late growth stages and increased BRA under HF. Baogu 19 showed more reduction amount on each vegetative tissue biomass than other cultivars （Table 3）. Therefore， the biomass reduction property in vegetative tissues during filling stage is regulated by fertilization and density and this trait shows drastic varietal difference among the tested cultivars. 　　Plant lodging resistant??associated traits in the cultivars under high fertility and high density treatments
　　A suite of lodging resistant??associated traits， including plant height （PH） and balancing position （BP）， and stem anti??broken resistance （ABR）， anti??puncturing resistance （APR）， stem length （SL）， stem diameter （SD） and lignin content of stem， were assessed at mid??filling stage. Among the cultivation treatments， HD had higher PH and BP， followed by HF and RC （Table 4）. Among the cultivars， Jigu 19 showed higher PH and BP， followed by 60D， Baogu 19， and 9050 （Table 4）. Stem ABR and APR were shown to be the highest in HF， followed by those in RC and HD. Among the cultivars， Baogu 19 showed the highest ABR and APR， followed by Jigu 19， 60D and 9050 （Table 4）. Stem lignin histochemical staining results are consistent with the ABR and APR behaviors （Fig. 1）. These results together indicated that high fertility and suitably lowered plant density can improve the lodging resistant??associated traits. Baogu 19 exhibited improved lodging resistant??associated traits under various cultivation treatments especially under high density condition， suggesting that its cultivation is relative less risk under lodging feasible cultivation condition.
　　Correlation analysis between stem anti??broken resistance （ABR）， a potential marker reflecting plant lodging resistance with other lodging??associated traits was performed. Results indicated that stem ABR is negatively correlated with PH， BP， SL， BP/PH ratio and positively correlated with APR and SD， showing positively significant correlation between ABR with APR and SD （Table 5）. These results indicated that ABR and stem lignin content impact largely on the lodging resistance capacity of millet plants at filling stage.
　　The yield formation traits and yields in the cultivars under different cultivation treatments
　　The spike weights， per??1 000 grain weights， and yields in tested cultivars under different cultivation treatments are shown in Table 6. Similar to plant biomass and biomass translocation amount as mentioned above， among the treatments， HF had increased spike weights， similar per??1 000 grain weights， and elevated yield compared with RC and HD. As to treatments RC and HD， HD showed increased yields but decreased spike weights and per??1 000 grain weights with respect to RC. However， a large variation was shown on yield among the cultivars， in which， Baogu 19 showed relatively high above traits compared with others under HD， suggesting its less high density inhibition on plant growth and yield formation. 　　Discussion
　　At late growth stage， the acquisition capacity of resources （e.g.， light， water， and indispensible inorganic nutrients） of plants and the internal resource translocation efficiency impact largely on the biomass production and biomass translocation property of vegetative tissues to harvest organ[19]. In millet and other cereal crops， the biomass assimilated in grain during seed filling are partly derived from vegetative tissue storage prior to flowering， which is transported into seed， a sink during filling stage[20-21]. In addition to genetic effects， cultivation techniques such as inorganic nutrient supplies （i.e.， N， P， and K） exert drastic roles in regulating plant biomass production and biomass partitioning of the vegetative tissues to seeds[23-24]. Compared with plants grown in rich soil， the crop plants cultivated in infertile soil with limited inorganic nutrients， such as N， exhibit largely reduction on biomass and yields， although they possess strengthened translocation rate of vegetative tissue biomass to seed[25-26]. In this study， using four summer millet cultivars as materials， we evaluated the increased fertilization treatment （with coordinately increase of N， P， and K supply） in regulating plant biomass production at flowering and maturity stages and biomass translocation property of vegetative tissues during seed filling stage. Similar to previous reports， the millet cultivars displayed large increase on plant biomass at both flowering and maturity stages， increased biomass translocation amount from vegetative tissues to seed during filling stage and improved yield， although comparable biomass translocation rates were observed in cultivars between the high fertility （HF） treatment and the regular culture （RC） treatment. Moreover， the increased plant biomass in cultivars at both flowering and maturity stages obtained under HF relative to RC， indicating that coordinately increased inorganic nutrients can promote the plant biomass production， vegetative tissue biomass translocation amount at late stage， and the yield formation in summer millet in North China.
　　Plant density acts as a critical cultivation technique in regulating biomass production and the yield formation potential[14]. In millet and other cereal crops， the effects of density on plant biomass and yields are genetic background??associated. Although the biomass at plant level are lowered along with the increase of densities， distinct cultivars exhibit to be much suitable to high density and obtain improved plant growth and yield formation in higher density ranges[13]. In this study， compared with RC， the plants of cultivars cultured under high density （HD） reduced the biomass at flowering and maturity stages at plant level， but obviously enhanced the biomass at above stages at planting area level， which led to the improved yields in cultivars examined. In addition， Baogu 19 exhibited increased biomass at late growth stages at both plant and planting area levels under HD compared with other cultivars， suggesting its potential in cultivation under HD condition given the high density tolerance. 　　Alleviation of plant lodging at late growth stages is a large benefit in cultivating millet and other cereal crops. The plant lodging behaviors and the lodging resistant??associated traits have been investigated. Genetic background[27-29] and environment factors such as cultivation practice exert huge effects on plant lodging behaviors. Previous， a set of plant lodging??resistant indices， including biomass distribution characteristic between shoot and root[30]， safety factors[29]， aerodynamics regarding wind??induced plant bending moment[11]， and spatial and temporal non??uniformity changes in plant population[29]， were proposed for evaluating the plant lodging resistance. In this study， we assessed several lodging resistant??associated traits and content of stem lignin， the secondary cell wall deposit of vascular tissues involving plant lodging under abiotic stresses[31-32]. The lodging resistant??associated traits were shown a drastic variation in cultivars under the cultivation treatments. Stem anti??broken resistance （ABR） was shown to be closely consistent with the stem lignin content and significantly correlated with stem diameter （SD） and plant balancing point （BP）， suggesting its valuable in predicting plant lodging resistance at late growth stage in millet.
　　Using integrated agronomic practices such as elite cultivar， balanced fertilizer input management， and suitable density can be effective in improving the crop productivity. In this study， our results indicated that suitably increased basal fertilizer supply and density together with elite cultivars， such as Baogu 19， can promote the plant biomass and the yield formation in summer millet cultivation in Hebei plain and North China.
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