Conversion of a normal maize hybrid into a waxy version using in vivo CRISPR/Cas9 targeted mutation activity

Waxy maize is a specialty maize that produces mainly amylopectin starch with special food or industrial values. The objective of this study was to overcome the limitations of wx mutant allele acquisition and breeding efficiency by conversion of parental lines from normal to waxy maize. The intended mutation activity was achieved by in vivo CRISPR/Cas9 machinery involving desired-target mutation of the Wx locus in the ZC01 background,abbreviated as ZC01-DTM^(wx). Triple selection was applied to segregants to obtain high genome background recovery with transgene-free wx mutations. The targeted mutation was identified, yielding six types of mutations among progeny crossed with ZC01-DTM^(wx).The amylopectin contents of the endosperm starch in mutant lines and hybrids averaged94.9%, while those of the wild-type controls were significantly(P < 0.01) lower, with an average of 76.9%. Double selection in transgene-free lines was applied using the Bar strip test and Cas9 PCR screening. The genome background recovery ratios of the lines were determined using genome-wide SNP data. That of lines used as male parents was as high as98.19% and that of lines used as female parents was as high as 86.78%. Conversion hybrids and both parental lines showed agronomic performance similar to that of their wild-type counterparts. This study provides a practical example of the efficient extension of CRISPR/Cas9 targeted mutation to industrial hybrids for transformation of a recalcitrant species.

Cob color, an indicator of grain dehydration and agronomic traits in maize hybrids

Screening for agronomic traits associated with grain moisture is important for mechanical grainharvesting of maize. Cob color as a visual indicator has received limited attention, though it has been subjected to artificial selection, and may have some association with threshability. To investigate the relationships between cob color and grain moisture and other agronomic traits, field experiments were conducted during 2016–2017 using 23 commercial hybrids with red or white cobs. Kernels of red-cob hybrids dehydrated faster, showing lower moisture content at harvest than white-cob hybrids. A cob color index(CCI) was established as a quantitative measure of cob color in the hybrid panel. Ranging from0(whitest) to 17.98(reddest), CCI correlated well with grain dehydration and other agronomic traits associated with growth ontogeny, plant morphology, and plant N content. Strong selection of red cob for recently released hybrids suitable for mechanical grain-harvesting indirectly validated the observed link between cob color and grain dehydration. We propose that cob color and CCI could be used in future selection of maize cultivars bred for mechanical grain-harvesting.

Development and application of marker-assisted reverse breeding using hybrid maize germplasm

Humankind has been through different periods of agricultural improvement aiming at enhancing our food supply and the performance of food crops. In recent years, whole genome sequencing and deep understanding of genetic and epigenetic mechanisms have facilitated new plant breeding approaches to meet the challenge of growing population, dwindling resources, and changing climate. Here we proposed a simple and fast molecular breeding method, marker-assisted reverse breeding（MARB）, which will revert any maize hybrid into inbred lines with any level of required similarity to its original parent lines. Since all the pericarp DNA of a hybrid is from the maternal parent, whereas one half of the embryo DNA is from the maternal parent and the other half from the paternal parent, so we firstly extract DNA from seed embryo and pericarp of a selected elite hybrid separately and then we derived the genotypes of the two parents with high-density single nucleotide polymorphism（SNP） chips. The following marker-assisted selection was performed based on an Illumina low-density SNP chip designed with 192 SNPs polymorphic between the two parental genotypes, which were uniformly distributed on 10 maize chromosomes. This method has the advantages of fast speed, fixed heterotic mode, and quick recovery of beneficial parental genotypes compared to traditional pedigree breeding using elite hybrids. Meanwhile, MARB has the advantage of not requiring sophisticated transformation and double haploid（DH） technologies over RNA interference（RNAi）-mediated reverse breeding. In addition, MARB can also be used with feed corn harvested from big farms, which is often similar to F_2 populations, and the relevant transgenes in the population can be eliminated by marker-assisted selection. As a result, the whole global commercial maize hybrids can be utilized as germplasm for breeding with MARB technology. Starting with an F_2 population derived from an elite hybrid, our experiment indicates that with three cycles of marker-assisted selection, selected lines could recover over 80% of the parental genotypes and associated beneficial genes in a fixed heterotic mode. The success application of MARB in maize suggests that this technology is applicable to any hybrid crop to breed new inbreds with improved hybrid performance but the same heterotic mode. As chip technology becomes cheap, it would be expected that polymorphism screening and following marker-assisted selection could be done with one all-purpose high density chip. Several issues associated with MARB were discussed, including its rationale, efficiency and advantages, along with food/feed and environmental safety issues and applications of MARB in variety protection and marker-assisted plant breeding.

Factors influencing hybrid maize farmers' risk attitudes and their perceptions in Punjab Province,Pakistan

Hybrid maize farmers have to face diverse kinds of climate, biological, price and financial risks. Farmers＇ risk perceptions and risk attitudes are essential elements influencing farm operations and management decisions. However, this important issue has been overlooked in the contemporary studies and therefore there is a dearth of literature on this important issue. The present research is therefore, an attempt to fill this gap. This study aims to quantify hybrid maize farmers＇ perceptions of disastrous risks, their attitudes towards risk and to explore the impacts of various farm and farm household factors on farmers＇ risk attitudes and risk perceptions. The present study is conducted in four hybrid maize growing districts of Punjab Province, Pakistan, using cross-sectional data of 400 hybrid maize farmers. Risk matrix and equally likely certainty equivalent （ELCE） method are used to rank farmers＇ perceptions of four catastrophic risk sources including climate, biological, price and financial risks and to investigate farmers＇ risk aversion attitudes, respectively. Furthermore, probit regression is used to analyze the determinants affecting farmers＇ risk attitudes and risk perceptions. The results of the study showed that majority of farmers are risk averse in nature and perceive price, biological and climate to be potential sources of risks to their farm enterprise. In addition, analysis divulges that distance from farm to main market, off-farm income, location dummies for Sahiwal and Okara, age, maize farming experience, access to extension agent, significantly （either negatively or positively） influence farmers＇ risk attitudes and risk perceptions. The study delivers valuable insights for farmers, agricultural insurance sector, extension services researchers and agricultural policy makers about the local understanding of risks to hybrid maize crop in developing countries, like Pakistan, and have implications for research on farmers＇ adaptation to exposed risks.

Effect of Fertilizing Level and Planting Densities on Yield and Nitrogen Utilization in Maize

Elite maize hybrid Guidan0810 was selected as material, and the effects of fertilizing level and planting densities on yield and nitrogen utilization were dis- cussed in the study. In field experiments as per double-cropping system, 4 main plots （fertilization levels） and 6 subplots （planting densities） were set in a split plot design. The results suggested that yield had close relationship with fertilization levels and planting densities. Different fertilization levels and planting densities significantly affected yield. With the increase of nitrogen fertilization, nitrogen use efficiency, nitrogen agronomic efficiency and nitrogen physiological efficiency declined. Under the same fertilization level, nitrogen use efficiency, nitrogen agronomic efficiency and nitrogen physiological efficiency grew a little with the increase of planting density, so nitrogen efficiency could be improved by regulating planting density. The results also showed that A2 （including N 225.0 kg/hm2, P205 75.0 kg/hm^2, K20 187.5 kg/hm^2） matching to B3 （52 500 plants/hm^2） or B4（60 000 plants/hm^2） was a better design, which could obtain a higher yield in the range of 7 913.2-8 207.8 kg/hm2, respectively.

Review of the detasseling techniques for maize(Zea mays L.)hybrid seed production

Maize(Zea mays L.)is a critical staple crop globally,integral to human consumption,food security,and agricultural product stability.The quality and purity of maize seeds,essential for hybrid seed production,are contingent upon effective detasseling.This study investigates the evolution of detasseling technologies and their application in Chinese maize hybrid seed production,with a comparative analysis against the United States.A comprehensive examination of the development and utilization of detasseling technology in Chinese maize hybrid seed production was undertaken,with a specific focus on key milestones.Data from the United States were included for comparative purposes.The analysis encompassed various detasseling methods,including manual,semi-mechanized,and cytoplasmic male sterility,as well as more recent innovations such as detasseling machines,and the emerging field of intelligent detasseling driven by unmanned aerial vehicles(UAVs),computer vision,and mechanical arms.Mechanized detasseling methods were predominantly employed by America.Despite the challenges of inflexible and occasionally overlooked,applying detasseling machines is efficient and reliable.At present,China’s detasseling operations in hybrid maize seed production are mainly carried out by manual work,which is labor-intensive and inefficient.In order to address this issue,China is dedicated to developing intelligent detasseling technology.This study emphasizes the critical role of detasseling in hybrid maize seed production.The United States has embraced mechanized detasseling.The application and development of manual and mechanized detasseling were applied later than those in the United States,but latest intelligent detasseling technologies first appeared in China.Intelligent detasseling is expected to be the future direction,ensuring the quality and efficiency of hybrid maize seed production,with implications for global food security.

On Significance of Heterotic Group Theory in Hybrid Rice Breeding

Heterotic group theory （HGT） has played a major role in supporting hybrid maize breeding for about 100 years. The basic content and studies of HGT, and its application in rice and maize were summarized in this paper. Additionally, difficulties and challenges for hybrid rice breeding in China were analyzed, and necessity and urgency in hybrid rice breeding by using HGT were proposed.

Comparative Study on Density Tolerance of Main Varieties in Southwest Maize Zone

This paper conducted a split-plot trial to study the yield,main agronomic characters and density tolerance of 11 maize varieties in southwest maize zone using the density and variety as experimental factors.The results indicated that ① the differences among density,variety and the intersection of density and variety were extremely significant;② Average yield of the tested varieties under high planting density（64 500 plant/hm^2） was significantly higher than that under low-density（49 500plant/hm^2）,and increased 10.37%;③ Four types were selected by the performance of all varieties under high planting density,such as high yield increase with high density,high yield but not increase with high density,low yield increase with high density,low yield but not increase with high density.

Decomposition of Surface-Applied and Soil-Incorporated Bt Maize Leaf Litter and Cry1Ab Protein During Winter Fallow in South Africa

Unintended effects of genetic modification on chemical composition of Bt maize leaf litter may have impacts on its decomposition. In most agricultural systems in South Africa, maize litter is either left on the soil surface or incorporated into the soil during tillage. A litterbag experiment, using leaf litter of three maize hybrids （DKC80-12B, DKC80-10 and DKC6-125）, was carried out at the University of Fort Hare Research Farm, South Africa, to determine the effects of genetic modification on decomposition of maize leaf litter when left on the soil surface under field conditions between July and November, the normal fallow period, in 2008. Another litterbag experiment was conducted at the University of Fort Hare Research Farm and Zanyokwe Irrigation Scheme, South Africa, using leaf ~itter of two maize hybrids genetically modified with the erylAb gene （MONS10）, DKC75-15B and PAN6Q-3OSB, and their corresponding near isolines, CRN3505 and PAN6Q-121. The degradation of CrylAb protein in the litter, both surface-applied and soil-incorporated, was also investigated. Decomposition of Bt maize litter was similar to that of non-Bt maize litter both when applied on the surface and when incorporated into soil. Soil-incorporated litter, as well as its CrylAb protein, decomposed faster than that applied on the surface. The leaf litter C：N ratios of PAN6Q-308B and PAN6Q-121 were similar throughout the study, whereas those of DKC75-15B and CRN3505 declined by similar amounts during a 12-week period. These findings suggested that decomposition of leaf litter of Bt maize, with the MON810 event, was not affected by maize genetic modification, and that the CrylAb protein broke down together with plant leaf litter during the winter fallow regardless of whether the litter was applied on the soil surface or incorporated into soil.

A Computational Workflow to Identify Allele-specifc Expression and Epigenetic Modifcation in Maize

Allele-specific expression refers to the preferential expression of one of the two alleles in a diploid genome, which has been thought largely attributable to the associated cis-element variation and allele-specific epigenetic modification patterns. Allele-specific expression may contribute to the heterosis （or hybrid vigor） effect in hybrid plants that are produced from crosses of closely-related species, subspecies and/or inbred lines. In this study, using Illumina high-throughput sequencing of maize transcriptomics, chromatic H3K27me3 histone modification and DNA methylation data, we developed a new computational framework to identify allele-specifically expressed genes by simul- taneously tracking allele-specific gene expression patterns and the epigenetic modification landscape in the seedling tissues of hybrid maize. This approach relies on detecting nucleotide polymorphisms and any genomic structural variation between two parental genomes in order to distinguish pater- nally or maternally derived sequencing reads. This computational pipeline also incorporates a mod- ified Chi-square test to statistically identify allele-specific gene expression and epigenetic modification based on the Poisson distribution.