Transgenic crops for the agricultural improvement in Pakistan

Transgenic technologies have emerged as a powerful tool for crop improvement in terms of yield, quality, and quantity in many countries of the world. However, concerns also exist about the possible risks involved in transgenic crop cultivation. In this review, literature is analyzed to gauge the real intensity of the issues caused by environmental stresses in Pakistan. In addition, the research work on genetically modified organisms (GMOs) development and their performance is analyzed to serve as a guide for the scientists to help them select useful genes for crop transformation in Pakistan. The funding of GMOs research in Pakistan shows that it does not follow the global trend. We also present socio-economic impact of GM crops and political dimensions in the seed sector and the policies of the government. We envisage that this review provides guidelines for public and private sectors as well as the policy makers in Pakistan and in other countries that face similar environmental threats posed by the changing climate.


Recent advancements in biotechnology resulted in rapid adoption of genetically modified (GM) crops in the agriculture systems. At the same time, transgene escape has also been reported and examples reveal global dimension of the problem. Pollen mediated gene flow (PMGF) is the major pathway for transgene escape. Almost all transgenes have been escaped into their Non-GM counterpart and wild relatives. Although gene flow varies between species, crops, and ecological zones/environments but intraspecific gene flow (> 10%) is not uncommon in adjacent populations. Whereas in outcrossing species, 1% gene flow at thousand meters’ isolation is not unusual, and magnitude is even higher than the mutation rate. It is well documented that transgene flow is deteriorating different production systems in agriculture and famers choice to cultivate GM, conventional and organic crops. If comprehensive policy is not implemented, then in future it will be difficult to detect and remove transgenes from the environment; if unexpected problems arise.

Genetic effects conferring heat tolerance in upland cotton (Gossypium hirsutum L.)

Cotton belongs to family Malvaceae containing more than
200 genera and about 2 300 species. There are more than
50 species of Gossypium reported till now, which are native
to Africa, Australia, Central and South America and Asia,
respectively (Fryxell 1992; Wendel and Grover 2015). Out
of 50 species, only four are domesticated and widespread.
Two diploid (2n = 26) species, namely G. arboreum and G.
herbaceum belong to Old World cotton produce only 1%
of the total cotton production in the world, whereas two
tetraploid (2n = 52) species, namely G. barbadense and G.
hirsutum belong to New World cotton produce 94% of the
total world cotton production. G. barbadense produces 4%,
while G. hirsutum also known as upland cotton produces
about 90% of the total cotton production in the world (Lu
et al. 1997; McCarty et al. 2004).
Upland cotton is a key source of spinnable fiber and cultivated
in more than 61 countries in the world on an area of
29.3 million hectares (ICAC 2018). Cotton and cottonbased
industry has a pivoting role in the economy of
Pakistan. Pakistan ranks the fourth in terms of area and
production in the world after India, China and USA, 3rd in
consumption and 2nd in yarn production in the world.
Cotton contributes 1% share in GDP, while 55% in total foreign
exchange earnings of Pakistan. Cotton was planted on
an area of 2.7 million hectares in 2017, showing an increase
of 10% over the previous year. About 8% more cotton production,
i.e., 11.54 million bales was recorded during 2017/
2018 as compared with 2016/2017 where 10.72 million
bales was recorded (PCCC 2017). However, in terms of per
acre yield (679 kg·hm− 2), Pakistan is lagging far behind
from the major cotton producing countries like Australia
(1 816 kg·hm− 2), China (1 719 kg·hm− 2), Turkey (1 826
kg·hm− 2) and USA (985kg·hm− 2) (ICAC 2018).
A loss of about one-third of cotton produce was recorded
in Pakistan during 2015/2016 due to adverse climatic conditions
particularly heavy rains during reproductive phase
of crop. But high temperature with dry weather conditions
favored the spread of whitefly in 2016 and 2017 which affected
the productivity of cotton crop on a wide range of
area in Punjab province. In recent times besides drought,
salinity, insect pests, diseases and seed quality: high
temperature has emerged as a major threat to cotton productivity.
It is estimated that the global temperature is increasing
by 0.4~0.8 °C/year (PMD 2016). The consequences
of high temperature in cotton could be low germination,
higher fruit shedding (≥ 30 °C/22 °C), pollen sterility and
abortion (Guilioni et al. 1997; Ismail and Hall 1999), unavailability
of macro and micro nutrients due to increase in
soil pH, higher levels of CO2 in the air will increase photosynthetic
activity resulting in enhanced nutrient requirement
of cotton plants.

Role of SNPs in determining QTLs for major traits in cotton

A single nucleotide polymorphism is the simplest form of genetic variation among individuals and can induce
minor changes in phenotypic, physiological and biochemical characteristics. This polymorphism induces various
mutations that alter the sequence of a gene which can lead to observed changes in amino acids. Several assays
have been developed for identification and validation of these markers. Each method has its own advantages and
disadvantages but genotyping by sequencing is the most common and most widely used assay. These markers are
also associated with several desirable traits like yield, fibre quality, boll size and genes respond to biotic and abiotic
stresses in cotton. Changes in yield related traits are of interest to plant breeders. Numerous quantitative trait loci
with novel functions have been identified in cotton by using these markers. This information can be used for crop
improvement through molecular breeding approaches. In this review, we discuss the identification of these markers
and their effects on gene function of economically important traits in cotton

Down regulation of cotton GbTRP1 leads to accumulation of anthranilates and confers resistance to Verticillium dahliae

Journal of Cotton Research

[Background] Verticillium wilt, caused by Verticillium dahliae, is called a “cancer” disease of cotton. The discovery and identification of defense-related genes is essential for the breeding of Verticillium wilt-resistant varieties. In previous research we identified some possible broad-spectrum resistance genes. Here, we report a tryptophan synthesis-related gene GbTRP1 and its functional analysis in relation to the resistance of cotton to V. dahliae.

[Results] Expression analysis shows that GbTRP1 is suppressed at 1 h and 6 h post V. dahliae infection, but activated at 12 h and 24 h, and the expression of GbTRP1 is highly induced by treatment with salicylic acid and jasmonic acid. Sub-cellular localization studies show that GbTRP1 is localized in the chloroplast. Suppression of GbTRP1 expression leads to lesion-mimic phenotypes and activates the immune response in cotton by showing enhanced resistance to V. dahliae and B. cinerea. Metabolomic analysis shows that anthranilic compounds significantly accumulated in GbTRP1-silenced plants, and these metabolites can inhibit the growth of V. dahliae and B. cinerea in vitro.

[Conclusions] Our results show that suppression of GbTRP1 expression dramatically activates the immune response and increases resistance of cotton to V. dahliae and B. cinerea, possibly due to the accumulation of anthranilate compounds. This study not only provides genetic resources for disease resistance breeding, but also may provide a basis for new chemical control methods for combatting of fungal disease in cotton.

[Title] Down regulation of cotton GbTRP1 leads to accumulation of anthranilates and confers resistance to Verticillium dahliae

[Authors] MIAO Yuhuan, ZHU Longfu and ZHANG Xianlong

Journal of Cotton Research. 2019; 2:19

Identification and Functional Analysis of Betv1 in Three D Genome-containing Wild Cotton Varieties


[Objective] By analyzing the major birch pollen allergen Betv1 gene family in D genomes of cotton and comparing the expression patterns of three diploid D-genome cotton varieties with different Verticillium wilt resistance levels, we aimed to provide a theoretical basis for further studies on the role of Betv1 genes in cotton resistant to Verticillium wilt. [Method] The Betv1 genes were identified, and a bioinformatics analysis of the physicochemical properties of their encoded sequences in Gossypium raimondii (D5) was performed. The transcriptome sequencing and quantitative real time-PCR of G. raimondii (D5), Gossypium trilobum (D8) and Gossypium thurberi (D1) were used to verify the expression patterns of Bet v 1 genes under Verticillium dahlia infection stress. Betv1 genes were silenced by virus-induced gene silencing in G. hirsutum to identify their functions. [Result] The D genome of cotton contains 59 members, 58 of which have introns and are distributed on eight chromosomes, and most encode hydrophilic proteins that localize to the cytoplasm. The expression levels of Betv1 genes in three wild cotton species having D genomes after being inoculated with V. dahliae were consistent with their disease resistance levels. The genes were separated into three groups based on their expression levels. Genes of Group 3 responded to V. dahliae infection and were highly expressed in disease-resistant cotton species G. thurberi. This indicated that Group 3 genes may be involved in the immune response of Verticillium wilt. A gene with a high expression level was screened out of Group 3. A corresponding homologous gene was silenced in G. hirsutum by virus-induced gene silencing, and gene-silenced plants were more susceptible to V. dahliae, indicating that the gene plays a positive regulatory role in the progress of Verticillium wilt resistance in cotton. [Conclusion] The Betv1 genes act in response to V. dahliae infection and are critical in cotton resistance to Verticillium wilt. The information obtained provides a basis for further studies of the cotton Bet v 1 family genes and their functions.

Key words: wild cotton; Verticillium wilt; Betv1 gene; transcriptome; quantitative real time-PCR (qRT-PCR); virus-induced gene silencing (VIGS)

Cotton Science. 2019, 31(5):361-380.

Comparative transcriptional analysis provides insights of possible molecular mechanisms of wing polyphenism induced by postnatal crowding in Aphis gossypii

Aphis gossypii is a worldwide sap-sucking pest with a variety of hosts and a  vector of more than 50 plant viruses. The strategy of wing polyphenism, mostly resulting from population density increasing, contributes to the evolutionary success of this pest. However, the related molecular basis remains unclear. Here, we identified the effects of postnatal crowding on wing morph determination in cotton aphid, and examined the transcriptomic differences between wingless and wing morphs.

Effect of postnatal crowding on wing determination in A. gossypii was evaluated firstly. Under the density of 5 nymphs·cm− 2, no wing aphids appeared. Proportion of wing morphs rised with the increase of density in a certain extent, and peaked to 56.1% at the density of 20 nymphs·cm− 2, and reduced afterwards. Then, transcriptomes of wingless and wing morphs were assembled and annotated separately to identify potentially exclusively or differentially expressed transcripts between these two morphs, in which 3 126 and 3 392 unigenes annotated in Nr (Non-redundant protein sequence) database were found in wingless or wing morphs exclusively. Moreover, 3 187 up- and 1 880 down-regulated genes were identified in wing versus wingless aphid. Pathways analysis suggested the involvement of differentially expressed genes in multiple cellular signaling pathways involved in wing morphs determination, including lipid catabolic and metabolism, insulin, ecdysone and juvenile hormone biosynthesis. The expression levels of related genes were validated by the reverse transcription quantitative real time polymerase chain reaction (RT-qPCR) soon afterwards.

The present study identified the effects of postnatal crowding on wing morphs induction and demonstrated that the critical population density for wing morphs formation in A. gossypii was 20 nymphs·cm− 2. Comparative transcriptome analysis provides transcripts potentially expressed exclusively in wingless or wing morph, respectively. Differentially expressed genes between wingless and wing morphs were identified and several signaling pathways potentially involved in cotton aphid wing differentiation were obtained.


JI Jichao, ZHANG Shuai, LUO Junyu, WANG Li, ZHU Xiangzhen, ZHANG Kaixin, ZHANG Lijuan & CUI Jinjie

Journal of Cotton Research. 2019,2:17

Genome-wide identification and expression analysis of DNA demethylase family in cotton

Journal of Cotton Research

[Background] DNA methylation is an important epigenetic factor that maintains and regulates gene expression. The mode and level of DNA methylation depend on the roles of DNA methyltransferase and demethylase, while DNA demethylase plays a key role in the process of DNA demethylation. The results showed that the plant’s DNA demethylase all contained conserved DNA glycosidase domain. This study identified the cotton DNA demethylase gene family and analyzed it using bioinformatics methods to lay the foundation for further study of cotton demethylase gene function.

[Results] This study used genomic information from diploid Gossypium raimondii JGI (D), Gossypium arboreum L. CRI (A), Gossypium hirsutum L. JGI (AD1) and Gossypium barbadebse L. NAU (AD2) to Arabidopsis thaliana. Using DNA demethylase genes sequence of Arabidopsis as reference, 25 DNA demethylase genes were identified in cotton by BLAST analysis. There are 4 genes in the genome D, 5 genes in the genome A, 10 genes in the genome AD1, and 6 genes in the genome AD2. The gene structure and evolution were analyzed by bioinformatics, and the expression patterns of DNA demethylase gene family in Gossypium hirsutum L. were analyzed. From the phylogenetic tree analysis, the DNA demethylase gene family of cotton can be divided into four subfamilies: REPRESSOR of SILENCING 1 (ROS1), DEMETER (DME), DEMETER-LIKE 2 (DML2), and DEMETER-LIKE3 (DML3). The sequence similarity of DNA demethylase genes in the same species was higher, and the genetic relationship was also relatively close. Analysis of the gene structure revealed that the DNA demethylase gene family members of the four subfamilies varied greatly. Among them, the number of introns of ROS1 and DME subfamily was larger, and the gene structure was more complex. For the analysis of the conserved domain, it was known that the DNA demethylase family gene member has an endonuclease III (ENDO3c) domain.

[Conclusions] The genes of the DNA demethylase family are distributed differently in different cotton species, and the gene structure is very different. High expression of ROS1 genes in cotton were under abiotic stress. The expression levels of ROS1 genes were higher during the formation of cotton ovule. The transcription levels of ROS1 family genes were higher during cotton fiber development.

[Title]Genome-wide identification and expression analysis of DNA demethylase family in cotton

[Authors] YANG Xiaomin, LU Xuke, CHEN Xiugui, WANG Delong, WANG Junjuan, WANG Shuai, GUO Lixue, CHEN Chao, WANG Xiaoge, WANG Xinlei & YE Wuwei *

Journal of Cotton Research. 2019, 2: 16

An isopentyl transferase gene driven by the senescence-inducible SAG12 promoter improves salinity stress tolerance in cotton

Journal of Cotton Research

[Background] Soil salinity seriously affects cotton growth, leading to the reduction of yield and fiber quality. Recently, genetic engineering has become an efficient tool to increase abiotic stress tolerance in crops.

[Results] In this study, isopentyl transferase (IPT), a key enzyme involved in cytokinin (CTK) biosynthesis from Agrobacterium tumefaciens, was selected to generate transgenic cotton via Agrobacterium-mediated transformation. A senescence-inducible SAG12promoter from Arabidopsis was fused with the IPT gene. Ectopic-expression of SAG12::IPT significantly promoted seed germination or seedling tolerance to salt stress. Two IPTtransgenic lines, OE3 as a tolerant line during seed germination, and OE8 as a tolerant line at seedling stage, were selected for further physiological analysis. The data showed that ectopic-expression of SAG12::IPT induced the accumulation of CTKs not only in leaves and roots, but also in germinating seeds. Moreover, ectopic-expressing IPT increased the activity of antioxidant enzymes, which was associated with the less reactive oxygen species (ROS) accumulation compared with control plants. Also, ectopic-expression of IPT produced higher K+/Na+ ratio in cotton shoot and root.

[Conclusions] The senescence-induced CTK accumulation in cotton seeds and seedlings positively regulates salt stress partially by elevating ROS scavenging capability.

[TitleAn isopentyl transferase gene driven by the senescence-inducible SAG12 promoter improves salinity stress tolerance in cotton

[Authors] SHAN Yi, ZHAO Peng, LIU Zhao, LI Fangjun* & TIAN Xiaoli

Journal of Cotton Research. 2019, 2: 15

Genetic analysis of yield and fiber quality traits in upland cotton (Gossypium hirsutum L.) cultivated in different ecological regions of China

Journal of Cotton Research

[Background] Cotton is an important fiber crop worldwide. The yield potential of current genotypes of cotton can be exploited through hybridization. However, to develop superior hybrids with high yield and fiber quality traits, information of genetic control of traits is prerequisite. Therefore, genetic analysis plays pivotal role in plant breeding.

[Results] In present study, North Carolina II mating design was used to cross 5 female parents with 6 male parents to produce 30 intraspecific F1 cotton hybrids. All plant materials were tested in three different ecological regions of China during the year of 2016–2017. Additive-dominance-environment (ADE) genetic model was used to estimate the genetic effects and genotypic and phenotypic correlation of yield and fiber quality traits. Results showed that yield traits except lint percentage were mainly controlled by genetic and environment interaction effects, whereas lint percentage and fiber quality traits were determined by main genetic effects. Moreover, dominant and additive-environment interaction effects had more influence on yield traits, whereas additive and dominance-environment interaction effects were found to be predominant for fiber traits. Broad-sense and its interaction heritability were significant for all yield and most of fiber quality traits. Narrow-sense and its interaction heritability were non-significant for boll number and seed cotton yield. Correlation analysis indicated that seed cotton yield had significant positive correlation with other yield attributes and non-significant with fiber quality traits. All fiber quality traits had significant positive correlation with each other except micronaire.

[Conclusions] Results of current study provide important information about genetic control of yield and fiber quality traits. Further, this study identified that parental lines, e.g., SJ48–1, ZB-1, 851–2, and DT-8 can be utilized to improve yield and fiber quality traits in cotton.

[Title] Genetic analysis of yield and fiber quality traits in upland cotton (Gossypium hirsutum L.) cultivated in different ecological regions of China

[Authors] SHAHZAD Kashif+, LI Xue+, QI Tingxiang, GUO Liping, TANG Huini, ZHANG Xuexian, WANG Hailin, ZHANG Meng, ZHANG Bingbing, QIAO Xiuqin, XING Chaozhu* & WU Jianyong*

Journal of Cotton Research. 2019, 2: 14