Transgenic crops for the agricultural improvement in Pakistan: a perspective of environmental stresses and the current status of genetically modified crops

ABSTRACT

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.

1. Introduction

Pakistan is a developing country with its economy highly dependent upon the agriculture. Agriculture sector contributes 18.86% to its GDP.¹ On an average, 5% increase in GDP per year has been recorded since 2005 along with the population explosion, especially in the rural areas, which are mostly affiliated with agriculture.² Biotechnology coupled with new breeding technologies exhibits the ultimate potential for genetic manipulation of agronomically and environmentally important crops. Cash crops which are paramount to acquire enhanced food production, quality improvement, and ensure food security are also there to guarantee an uplifting of the national economy. Tree biotechnology has also attracted researchers as climate change demands developing traits which were traditionally not present in trees. Additionally, biotechnological improvement is also important for trees due to absence of cross breeding owing to long juvenile periods. Besides, various diseases are also needed to be cured through genetic transformation as other control measures do not seem effective in the changing climate scenario.^3–5 In 1985, modern biotechnology was first implemented in Pakistan and by now 56 (50 in public sector and 6 in private sector) high-tech research institutes of biotechnology have been founded throughout the country, and majority of them work on the development of potential genetically modified (GM) crops with the properties that would help them fight against both biotic and abiotic environmental stresses.^6,7

Bt cotton event Mon-531 which carries Cry1Ac gene is the only transgenic crop recommended for general cultivation in Pakistan.⁸ Though 56 institutes are counted, working on developing transgenic plants in the country, but except a few, their ages are too less to give a thoroughly tested transgenic event for general cultivation. As Pakistan has been a signatory of Cartagena Protocols on Biosafety, therefore, National Biosafety guidelines were approved under Biosafety rules during 2005 to regulate research on development and commercialization of GM crops. A three-tier system was developed following these guidelines and National Biosafety Committee was taken as the apex body to finally permit the cultivation of genetically modified organisms (GMOs). This resulted in regularized cultivation of Mon-531 in the country in 2010. Eighteenth amendment was done in the constitution the same year that brought gross administrative changes in the governmental set-up and the system has been at halt since then. Environmental Protection Agency that was under Ministry of Environment became provincial subject, that generated ambiguity in the bureaucracy as biosafety rules were developed as obligation to Cartagena protocols which were signed by the federal government. An attempt was made to resolve the issue by introducing biosafety act in 2015 that declared National Biosafety Committee as Federal subject and brought its subsidiary committees under Ministry of Climate Change at provincial level. This decision was then challenged in the court by a body of farmers, hence the system is still at halt and many projects are waiting for field and/or lab experiments and the products are shelved.^9–12

Pakistan started investing heavily in scientific research by means of establishment of the Higher Education Commission (HEC) in 2002. Higher Education Commission has been sending scholars abroad for PhD studies and has funded many existing local universities and degree-awarding institutions, and has helped establish many new universities at the same time. Participants of PhD study abroad programs started returning back to Pakistan after completion of 4–5 years of studies in foreign universities and by now thousands of them are implementing their research programs locally. A great number out of them are trained in GMOs development. They are getting funded by HEC and some other national and international funding agencies.^13,14 In order to get maximum benefits from these investments, it is of principle importance to guide the new researchers through literature about the selection of genes, those are the most likely to improve agronomic performance of crop plants. This review explores the literature on the existing issues of agriculture in Pakistan, looks at the transgenes that have been developed either in the field or lab conditions, and also analyses transgenic events commercialized so far and then reviews projects funded by several funding agencies. This literature will help the scientists to better design their future experiments and maximize improvements needed for agronomic applications using transgenic crops.

2. Issues and Challenges for Yield Sustainability of Major Crops in Pakistan

Pakistan is located in the sub-tropical and semi-arid regions of the globe where variation in precipitation is significantly high during different seasons i.e. up to 250 mm (10 inches) per year in the North (Himalayas, Kashmir, and Skardu), up to 180 mm (7 inches) per year in desert or semi-desert regions of the South (Hyderabad and Turbat), up to 450 mm (17.7 inches) per year in Khyber Pakhtoon Khwa (Peshawar), 510 mm (20 inches) in Punjab (Lahore), and exceeds 500 mm (60 inches) in Abbottabad.^15,16 This results in conditions like drought, water logging, and salinity in many areas of country quite frequently.¹⁷ These stresses are divided into various categories as discussed below.

Figure 1.

Production Statistics of Major Crops of Pakistan (2000–2018).

2.1. Salinity and Sodicity

The total area of Pakistan is 79.6 million hectares; 23.8 million hectares are engaged in cultivation which is approximately 30% of the total land area of the country.¹⁸ Sodicity and salinity are the two major challenges for agriculture in Pakistan. The issue is more serious in climatic regions where irrigation is insufficient (10% of total land). Regions where human-induced soil erosion leads to different types of salts accumulation in soil also bear challenges associated with sodicity and salinity.¹⁹ It has been reported that 0.04 million hectares of Pakistan’s fertile lands are getting saline every year because of blatant floods, imbalance rainfall; most of this land belongs to Sindh province. So far, an area of 5.33 million hectares has been affected by this issue which has led up to 50% reduction in crops yields with an ultimate loss of $475million per annum.^20,21

The limitations of crop production and consequent negative effects are caused by salinity. Different salinity-tolerance levels have been observed in different crops i.e. maize, wheat, cotton, sugarcane, and rice which ultimately lead to yield reduction at different levels for each of the crops. The loss of production of these crops is analyzed under saline conditions and certain approximations like the level of electrical conductivity (EC) are monitored. Soil EC at which crop yield starts decreasing is termed as threshold EC. Among crops grown in Pakistan, cotton is found to be the most salinity-tolerant crop as its threshold EC level is 9.6, 13, and 17 dS/m (Deci Siemens per meter) at 10%, 25%, and 50% reduced production, respectively. On the contrary, maize and sugarcane are comparatively less salinity-tolerant crops. Table 1 shows the loss in crop yields due to salinity stress in various crops.

Table 1.

Yield loss in crops by salt-stress.

		EC (dS m−1) at which yield decreases by
Crop	EC threshold (dS m−1 at 25°C)	10%	25%	50%
Maize	1.7	2.5	3.8	5.9
Cotton	5.9	9.6	13.0	17.1
Rice	4.0	4.6	5.8	7.4
Sugarcane	1.8	3.4	5.9	10.0
Wheat	5.0	7.4	9.5	13.0

Source: (Zaman and Ahmad, 2009)²²

The data of salinity-tolerance presented in Table 1 show the EC threshold of the major crops of Pakistan and the percentage loss in yield as per salinity level in soil. The data on salt-stress show that cotton is tolerant to relatively higher salinity levels in soil while maize exhibits the lowest tolerance against the salt stress. Transgenics for salinity-tolerance are needed more in cereals compared to cotton.

2.2. Drought and Heat Stress

Pakistan has been experiencing severe consequences of the global warming for over a decade seeing extreme temperatures and drought conditions which turn the arable lands to barren ones and which poses a potential threat to productivity and country’s economy.²³ Sardar et al.²⁴ reported the occurrence of drought caused by abnormally dry weather and insufficient precipitation over the land has resulted in the fertility loss as the water availability became limited due to reduced river flows and dried canals, particularly in Baluchistan and Sindh provinces. The estimated loss of $2.0 billion per annum has been reported for these disasters to agriculture.²⁵ Pakistan’s cultivated area of 15.0 million hectares has been abandoned due to drought conditions.²⁶

The reduction in crop yield caused by drought is more significant than all other stress factors. Numerous yield-related characteristics of crop plants such as leaf size, rate of transpiration, stem extension, soil microbial activity, and root proliferation are significantly affected by the drought stress.²⁷ Although heat stress has yet not been taken as a serious consideration in Pakistan as compared to other environmental stress factors which are the focus of scientists/breeders, still most of the drought-combating biochemical mechanisms remain the same for both drought and heat stresses.²⁸ Drought affects various physiological processes of plant negatively and ultimately curtails the yield. These adverse impacts on yield depend upon the stress severity and the growth stage of the plants. Significant yield losses in major field crops due to drought stress have been reported in Table 2. Rice is the most susceptible crop to drought and heat stresses which cause a yield loss of up to 92% and 50%, respectively. Similarly, a loss of 57% and 31% of yield has been noted in wheat due to drought and heat, respectively.

Table 2.

Yield loss in crops by drought and heat stress.

Species	Stress	Yield Loss (%)
Wheat	Drought Heat	57 31
Maize	Drought Heat	63–87 42
Rice	Drought Heat	53–92 50
Soybean	Drought	46–71
Chickpea	Drought	45–69
Sunflower	Drought	60

Source: (Fahad et al., 2017)²⁸

From the data presented above it could be concluded that drought tolerance is direly needed not only in cereals but also in pulses while heat tolerance is also a matter of a great concern in cereals which requires biotechnological interventions.

2.3. Irrigation and Waterlogging

Pakistan owns one of the largest irrigation systems in world but still its capacity is not sufficient to meet up the water demands for its agriculture. With the passage of time extensive/multiple cropping systems have been adopted to meet the needs of expanding population in Pakistan.²⁹ Inadequacy of water supply is a potential threat to the economy of Pakistan. This inadequacy of irrigation water supply, due to climate change, maximizes the probability of occurrence of extreme weather events like drought and floods.³⁰ Besides natural disasters, politics of neighboring country, India, is also playing a major role in the water crisis in Pakistan. India has been continuously violating Indus water treaty (IWT) mediated by the World Bank in 1960. It is estimated that Pakistan would suffer a loss of 31 million acre feet (MAF) of water by the year 2025.^31,32

Pakistan has experienced multiple severe floods since 1950s and their frequency has increased in the last decade as it started to hit millions of hectares of land every year causing such adverse after effects as saline and water-logged soils.^30,33 An area of 1.55 million hectares has been affected by waterlogging so far,²¹ and it has been reported that 0.1 million hectares are still being affected by it every year which greatly affects the crop yield and GDP accumulating to a loss of $300 million annually.^34,35 In the recent past, the decline in crop production and yield collection had been experienced as the result of decrease of the cultivated area due to continuous blatant flooding, water logging, and increasing soil salinity.

Different crops exhibit varying tolerance levels to waterlogging which therefore leads to a reduction in yield of each crop. The crops that are less tolerant to waterlogging get severe production losses. In contrast to this, the crops having waterlogging-tolerant characteristics get less reduction in production. Sugarcane is the crop most sensitive to the waterlogging as up to 58% reduction in its yield was observed at the water table depth of 0–1.0 m. Though cotton and wheat are relatively tolerant to waterlogging yet the yield losses of 52.75% and 46.25%, respectively, are reported (Table 3). A biotechnological intervention to increase resistance against water logging will be worthwhile in this case.

Table 3.

Yield loss in crops by waterlogging.

Water-Table Depth (m)	Cotton	Sugarcane	Wheat	Average
0.00–0.25	98	91	79	89.3
0.25–0.50	57	66	49	57.3
0.50–0.75	35	46	29	36.6
0.75–1.00	21	29	28	26
Average (0.00–1.00)	52.75	58	46.25	52.3

Source: (Zaman and Ahmad, 2009)²²

2.4. Weeds Infestation

Weeds are one of the major challenging issues encountered in Pakistani agriculture. Delayed harvesting, stunted plant growth, and reduced adulterated yields are the drawbacks of infestation of weeds.³⁶ The crop under weed stress grows in a competitive environment which ultimately leads to the yield loss of up to 40%. Reduced quality of produce due to the intermixing with the weed seeds further reduces the farmers’ income. Herbicides are applied to the crops for the weed control how ever crop plants may also get stressed by their application. The issue of whether the herbicides are in favor of or against the crop production still remains a matter of debate.³⁷ Selective herbicides are used for weed control in cultivated fields but they also leave the adverse effects on the crop plants. A loss of up to 998 million USD occurs every year due to the need to purchase of herbicides and due to the yield losses.³⁸ Hence, there is the need for the development of herbicide tolerant crops to make sure that crops would not get affected or damaged while the weeds would be targeted by the herbicide action. It is believed that weed control through biotechnological interventions might offer a significant help in coping with these challenges by acquiring selective resistance in crops via genetic engineering techniques. With the elimination of weeds, crops would have no more competition for nutrients and water. Hence, remarkable change in the crop yield could be achieved.³⁹

2.5. Insect Attack

Insect attack is the most crucial biotic stress to the plants in Pakistan and 25–75% of annual yield loss has been reported due to this issue. In order to overcome this drastic challenge to agriculture, certain advanced insect control approaches have to be implemented for the efficient insect pest control.⁴⁰ In Pakistan, 35–40% of the total yield of wheat is lost due to aphid attack and up to 50% loss in maize yield is reported due to pest attack.⁴¹ Rice is attacked by various insect species and faces loss of up to 37% annually, Potato bears 40% yield loss due to insect interference,⁴² while in cotton yield loss of up to 28% was reported during 2016 and the impact of yield loss by this factor to the Pakistan’s economy is more than one billion dollar only in cotton industry annually.^43,44 A broad range of insect pests greatly affect the crop productivity. To counter this situation, farmers and researchers are adopting different control practices such as synthetic/chemical insecticides. Since last three decades, the use of these chemicals is prevailing for crop protection, leaving adverse effects on the environment, human, and animal health.^42,45–47 The injudicious application of synthetic insecticides has caused the major outburst of different insect pests. In the recent decade, Integrated Pest Management (IPM) approach has been applied because of its environment friendly characteristics, long-lasting effects, and inexpensiveness.⁴⁸ The global emergence of insect-resistant crop varieties has developed the interest of researchers in crop improvement through transgenic strategies. In Pakistan Mon-531 transgenic event of transgenic Bt cotton has reduced the multiple applications of insecticides that help control the chewing insects.⁴⁹

3. Impacts of Climate Change and Pollution on the Crop Production

Biotic and abiotic stresses pose a serious threat to agriculture. The adverse global effects are expected to affect arable lands due to increased salinization which might lead to a loss of ~30% of entire cultivated land in next 25 years. And up to 50% might be lost by 2050 due to increased flooding and limited water availability to rain-fed lands as a result of climate change.⁵⁰ The future variations in production of maize, rice, and wheat in spring season for the US and Europe are projected by the two Representative Concentration Pathways (RCPs) of the Intergovernmental Panel on Climate Change (IPCC) i.e. RCP4.5 and RCP8.5, estimated for year 2050 relative to 2000 (Fig. 2). Ozone and Climate change are directly linked with each other; increase of ozone on earth surface results in more retention of heat, leading to high temperatures and disturbance of the other components of atmosphere resulting in severe climate change which affects agriculture negatively. Burning of Chlorofluorocarbons and higher temperatures at stratosphere has resulted in Ozone depletion giving access to Ultraviolet rays leading to global warming.

Figure 2.

Effects of ozone air pollution and climate change on crop production in different regions of the world.

Source: Tai et al. (2014; 2017)^51,52

4. Need for GMOs and Genes for Pakistan

Plant biotechnology plays a vital role in sorting out the major issues such as salinity and drought stresses in crops and trees for fulfilling the nutrition requirements and the maintenance of the natural ecosystem. The regulation of certain stress-responsive genes plays a significant role in controlling pathways related to abiotic stress tolerance at molecular level which involve proteins and membranes protection, cell signaling, scavenging of toxic compounds, free-radicals, etc. At present, the work on stress-responsive mechanisms has been found beneficial with certain genetic manipulations made to acquire stress tolerance which can be applied onto ecologically and agriculturally important crops.^53–57

Genetic manipulation of complex traits may cause difficulty in achieving abiotic stress tolerance efficiently in plants. Monogenic traits like ubiquitinous expression of antiporters and transcriptional factors seem to be relatively less difficult in achieving abiotic stress tolerance as several cases have been reported to be successful due to remarkable improvements in stress tolerance. On the contrary, the efforts are being made to develop stress-tolerant plants with the help of heterologous genes and stress tolerance-associated genes including ones from the organisms growing in the extreme environmental conditions i.e. thermophiles and halophytes.^58,59 The success of gene/s may depend upon their origin when the genetic modification of plants through the introduction of foreign genes is done. Numerous factors are needed to be accounted for while designing exogenous gene expression in plants which include environmental and human health considerations, diverse metabolites availability in plants, and different post-transcriptional or translation a modifications of foreign genes.^60–63

There are few ecological benefits that natural environment can offer while acquiring adaptation against some stress, this might get masked in agriculture due to energy and metabolic costs that cause yield reduction. Combined implementation of both traditional and molecular breeding can help in achieving abiotic stress tolerance in agriculturally important plants.^64–66 Therefore, compendious breeding strategy should involve germplasm selection, conventional breeding, biotechnology-oriented improvement of breeding and selection procedures, adaptation and improvement of agricultural practices, utilization of molecular markers and probes for selection purposes, genetic transformation and elucidation of regulatory mechanisms in sensitive and in tolerant genotypes for the purposes of inducing abiotic stress tolerance.^67–69

An inducible stress-responsive expression should be optimized in GM crops which should be highly regulated and which should not interfere with the metabolic machinery of the host plant under normal environmental conditions.⁶⁶ The evaluation of a few stress-resistant GM crops under stressed environmental conditions in field trials hasled to the removal of various transgenics from the research area.⁶⁵

Stress-tolerant plants which have been developed until now through genetic modifications are applied for both agronomic and horticultural crops i.e. wheat,⁷⁰ maize,⁷¹ rice,⁷² tobacco,⁷³ mustard,⁷⁴ soybean,⁷⁵ sugarcane,⁷⁶ cotton,⁷⁷ citrus,⁷⁸ eggplant,⁷⁹ grapes,⁸⁰ banana,⁸¹ and potato.⁸² Designing, testing, and commercialization of new stress-tolerant varieties have been found imperative at the stage of observation of the acquired abiotic stress tolerance.⁸³

The list of stress-responsive genes, both for biotic and abiotic stresses presented in Table 4 is meant to be taken into consideration during the development of a new stress-tolerant crop variety in Pakistan as these are the proven genetic entities that can be quite beneficial in the enhancement of the crop yields.

Table 4.

List of reported stress responsive genes.

Genes for Salinity-Tolerance
Genes	Proteins	Targeted Plants	Gene Description	Improvement (~%age)	References
AKT1 AKT2 KAT1	Ion Transporters	thaliana	Arabidopsis K+ Transporters		50
ALFIN1	Transcription Factor	A. thaliana	Alfalfa zinc finger protein		84
AVP1	Ion Transporter	A. thaliana	H+ Pyrophosphatase	19	85
CNGCs-GLR	Channel proteins	O. sativa	Cyclic nucleotide-gated channels Glutamate receptor-like		86
CYP709B3	Monooxygenase	A. thaliana	Cytochrome P450 family 709B	15	87
DREB2A	Transcription Factor	O. sativa	Dehydration responsive element-binding protein 2A	32	88
HAL1 HAL3	Stress responsive Protein	C. melo	Halotolerance Protein	17	89,90
HKT1	Ion Transporter	O. sativa	High affinity K+ transporter	28	91
IMT1	Transport catalyzing enzyme	N. Tabacum	Inositol 4-methyltransferase	18	92
MIPS	Biosynthetic enzyme	A. thaliana	Myo-inositol 1p-synthase I	45	93
NAC	Transcription Factor	O. sativa	Transcriptional Factor	30	94
NHX1	Ion Exchanger	A. thaliana	Na+/H+ Exchanger		95
SOS1	Transporter protein	A. thaliana	Salt overly sensitive/Na+/K+antiporter	26	96
SP1	Transcription Factor	Populus	Transcription Factor	20	97
TaSC	Stress responsive Protein	A. thaliana	Triticum aestivum salt-tolerant correlative	17	98
WRKY3	Transcription Factor	A. thaliana	DNA-binding protein	22	99
Genes for Drought Tolerance
ABF3 ABF4	Transcription Factor	O. sativa	ABA responsive element-binding factor	21	72
APX3	Antioxidant enzyme	O. sativa	Ascorbate peroxidase 3	18	100
BZIP46	Transcription Factor	O. sativa	Basic leucine zipper domain TF 46	19	101
COX1	Transmembrane protein	O. sativa	Cytochrome C oxidase subunit 1		102
CuZn-SOD Fe-SOD Mn-SOD	Antioxidant enzyme	Z. mays	Superoxide dismutase		103
CYP82A3	Monooxygenase	G. max	Cytochrome P450 82A3		104
DRAP1	Transcription Factor	O. sativa	Down regulator associated protein 1		105
GBF3	Transcription Factor	A. thaliana	G-box binding factor 3	22	106
HVA1	LEA protein	T. aestivum O. sativa	Hordeum vulgare ABA-inducible protein		107,108
Hahb-4	Transcription Factor	Helianthus	Helianthis annuus Homeobox-4		109
LEA3 LEA4	LEAs	O. sativa N. tabacum	Late embryogenesis abundant protein group 3,4	14	110,111
MDAR	Antioxidant enzyme	E. coracana	Monodehydroascorbatereductase		112
MYB2 MYC2	Transcription Factor	A. thaliana	Transcription Factor	60	113,114
NAC085	Transcription Factor	A. thaliana	NAC domain containing protein 85		115
P5CS	Biosynthetic enzyme	Petunia	Pyrroline carboxylate synthetase	18	116
PgTIP1	Aquaporin	A. thaliana	Panax ginseng tonoplast aquaporin		117
PIP	Aquaporin	Lactuca sativa	Plasma membrane intrinsic protein		118
Stpd1	Biosynthetic enzyme	Japanese persimmon	Sorbitol 6-phosphate dehydrogenase		119
Genes for Heat Tolerance
Hsa32	Stress responsive protein	A. thaliana	Heat stress associated protein 32		120
Hsp17.6A Hsp17.7 Hsp21 Hsp70-1	Stress responsive protein	A. thaliana Daucus Carota A. thaliana N. tabacum	Heat shock protein 17.6A Heat shock protein 17.7 Heat shock Protein 21 Heat shock Protein 70-1	25	121–124
HsfA2 HsfA3	Transcription Factor	A. thaliana	Heat stress transcription factor A2 Heat stress transcription factor A3	40	125
HSF1 HSF3	Transcription Factor	A. thaliana	Heat shock factor protein	30	126,127
Sp17	Transcription Factor	O. sativa	Spotted leaf gene		128
ZFP	Stress Responsive Protein	O. sativa	Zinc Finger Protein		129
FAD7	Dienoic Fatty Acids	O. sativa	Fatty Acid Desaturase-7	42	130
SBPase	Photosynthetic Enzyme	O. sativa	Sedoheptulose Phosphatase		131
GSK1	Enzyme	O. sativa	Glycogen Synthase Kinase-1		132
Genes for Cold Tolerance
CspA CspB	Cold responsive protein	Z. mays	Cold shock proteins		133
BetA	Biosynthetic Enzyme	Z. mays	Choline dehydrogenase		134
Cbf1 Cbf2 Cbf3 DREB	Transcription factor	Cucurbita pepo A. thaliana	C-repeat binding factor Dehydration responsive element binding factor		135,136
Cor15a Cor39	Cold responsive protein	N. tabacum T. aestivum	Cold regulated 15A Protein		137,138
GPAT	Biosynthetic Enzyme	Paeonia lactiflora Pall	Glycerol 3-phosphate acyltransferase		139
Gst Gpx	Stress responsive protein	Brassica Oleracea	Glutathione S transferase Glutathione peroxidase		140
Genes for Herbicide Tolerance
Aad-1 Aad-12	Herbicide responsive protein	Z. mays L.	Aryloxyalkanoate Dioxygenase	70	141,142
ACCase	Herbicide responsive protein	Avenafatua	Acetyl-coA carboxylase		143
AHAS (ALS) HrASurB	Biosynthetic enzyme	Cicer arietinum	Acetohydroxyacid synthase Acetolactate synthase		144
Bar Pat	Herbicide responsive protein Amino acid biosynthesis Inhibitor	N. tabacum T. aestivum	Bialaphos resistance Phosphinothricin acetyl transferase	9	145,146
Barstar	Ribonuclease inhibitor	Brassica	Barnaseribonuclease inhibitor	20	147
Bxn	Herbicide responsive enzyme	Trifoliumsubterraneum	Bromoxynil Nitrilase	38	148
Dmo	Herbicide responsive protein	Brassica scoparia	Dicamba monooxygenase	23	149
Epsps	Pathway Inhibitor	O. sativa	5-enolpyruvyl shikimate 3-phosphate synthase	40	150
GAT	Herbicide responsive protein	A. thaliana	Glyphosate N-acetyltransferase		151
GOX	Herbicide responsive protein	B. napus	Glyphosate oxidoreductase	9	152
PPO	Herbicide responsive protein	A. thaliana	Protoporphyrinogen oxidase	30	153
Csr1-2	Herbicide responsive protein	A. thaliana	Chlorsulfuron/imidazolinone resistant 1–2 Modified acetohydroxyacid synthase large subunit (AHASL)		154
Hppd	Herbicide responsive protein	G. max	Hydroxy phenylpyruvate Dioxygenase Inhibitor	30	155
Epsps and GAT	Coexpressed resistant proteins	G. max N. tabacum	Coexpression of EPSPS and GAT	20	156,157
Genes for Insect Resistance
Api	Inhibitor Protein	S. lycopersicum	Arrowhead Protease Inhibitor		158
CpTI	Inhibitor Protein	B. rapa	Cowpea Trypsin Inhibitor		159
Vip3A Vip3Aa20	Insect resistant Protein	Z. mays	Vegetative insecticidal protein 3 Aa20		160,161
Cry1A.105 Cry1Ab Cry1Ac Cry2Ab2 Cry3A Cry51Aa2 Cry3Bb1 Cry9C Cry34Ab1 Cry35Ab1 Cry1F	Insect-resistant toxins	Z. mays G. hirsutum Punica granatum	Crystalline toxins family	75	162–167
H12 H18 H24 H25	Insect-resistant proteins	T. aestivum	Hessian fly resistant gene family		168
Snf7	Insect-resistant protein	Z. mays	Vacuolar sorting protein		169
Genes for Pathogen/Disease Resistance
AC1	Inhibitor Protein	G. hirsutum	Replication associated protein		77
CMVcp	Pathogen resistant protein	C. annuum	Cucumber Mosaic Virus-Coat Protein		170
Lr28	Pathogen resistant protein	T. aestivum	Leaf rust 28		171
POX	Antioxidative enzyme	S. officinarum	Peroxidase		76
PAL	Pathogen resistant protein	N. tabacum	Phenyl-alanine ammonia lyase		172
PR2 PR5 PR10	Pathogen resistant protein	A. thaliana Prunus domestica T. aestivum	Pathogenesis related 2 Pathogenesis related 5 Pathogenesis related 10		173–175
CAD12 SAD	Plant defense protein	T. aestivum	Cinnamyl alcohol dehydrogenase 12 Sinapyl alcohol dehydrogenase		176
Rpi-vnt1	Pathogen resistant protein	S. tuberosum	Late blight resistance protein		177
Tlp	Pathogen resistant protein	T. aestivum	Thaumatin-like protein		178
Genes for Growth/Yield/Quality Improvement
Ccomt	Biosynthetic Enzyme	Medicago sativa	Caffeoyl CoA O-methyltransferase	30	179
CAld5H	CYP450-dependent Monooxygenase	A. thaliana	Coniferaldehyde 5 hydroxylase		180
Pgip	Inhibitory Protein	S. lycopersicum	Polygalacturonase Inhibitor Protein		181
Acc	Biosynthetic Enzyme	Pelargonium × hortrum	1-amino cyclopropane carboxylate synthase		182
Accd	Catabolic Enzyme	S. lycopersicum	1-amino cyclopropane carboxylate deaminase		183
Anti-Efe	Inhibitory Protein	S. lycopersicum	Anti-ethylene forming enzyme		184
Sam-K	Catabolic Enzyme	Persea americana	S-adenosylmethionine hydrolase		185
Bbx-32	Transcription factor-interacting protein	G. max	B-Box-32 Protein	23	186
Crt1	Biosynthetic enzyme	O. sativa	Carotene desaturase (Calreticulin-1 precursor)		187
CrtB Psy	Biosynthetic Enzyme	Z. mays	Phytoene synthase		188
Ms26 Ms45	Biosynthetic Protein	Z. mays	Male fertility proteins		189

Table 4 offers a list of transgenes integrated in order to combat the stressed environmental conditions worldwide which include various antioxidative enzymes, aquaporins, inhibitor proteins, ion transporters, LEAs, metabolic enzymes, monooxygenases, resistant proteins, stress-responsive proteins, and transcriptional factors. Genes exhibiting optimal potential against particular stresses must be prioritized during the planning of transformation against relevant encountered stress in Pakistan.

Here in Table 4 at least 17 studies are reported for having achieved a tolerance against salt stress, mostly in model plants. Out of these studies the best performing genes against salt tolerance reported include MIPS, DREB2A, and NAC which have caused a significant improvement in salt tolerance i.e., up to 45%, 32%, and 30%, respectively.^88,93,94 Whereas, the outcomes obtained from transgene expression of HKT1, SOS1, and WRKY3 were also remarkable but had induced non-significant improvement in the targeted plants.^91,96,99 These genes can be considered for transformation of crops to be grown in flood-affected lands and areas that have soils with excessive salt levels. The 21 studies presented in Table 4 have revealed that such genes as MYB2, MYC2, GBF3, ABF3, and ABF4 have exhibited their involvement in increasing of the drought tolerance in transgenics with 60%, 22%, and 21% improvement relative to non-transgenics^{72,106,113,114} while the rest of them have inconclusive results. Heat-tolerant genes such as FAD7, HsfAs (HsfA2 and HsfA3), HSFs (HSF1 and HSF3), and Hsps facilitate the plant in carrying out metabolic functions even under the stressed conditions like drought and rise in temperature.^{124,125,127,130} Various transgenes for the cold-stress tolerance including Csps, Cbfs, Cors, and WCS120 from cold-regulating gene families have been reported to be transformed in Arabidopsis, maize, tobacco and other crops and show variable resistance against cold stress. Based on these assumptions it would be important to stress the need to explore these cold stress-responsive genes.^133,136,138

Much work has been done over inducing herbicide tolerance for the sake of weed management to avoid competitive growth of the plants. Certain genes like Aad1-2, Bxn, Epsps, Hppd, and Ppo are the most significantly performing transgenes that cause approximately 30–70% improvement in the transgenics depending on a specific concentration of dose. Most of the soybean grown in the world carries glyphosate resistance using EPSPS or Gox genes. Bar gene is used as a selectable marker gene for selecting glufosinate-resistant transgenic plants. Limited area is also under crops carrying Bar for resistance against herbicides like Basta, Liberty, etc. Most of these genes belong to different bacterial sources.^{141,148,150,153,155}

Crytoxins are the most commonly used insect-resistant proteins that are being used since last few decades for the purposes of integrated pest management with an application to cotton, soybean, and maize.^164–166 Research is also underway to identify genes to ensure resistance against insects that remain unaffected by Cry genes. Various genes for the development of disease-resistant plants have been reported. Those include Ac1, PAL, POX, and PR-proteins however statistics regarding improvements that they caused in crops not available yet^{76,77,172,173} Acc, Bbx32, Ccomt, Ms, and many other genes for improving crop yield and quality have been successfully expressed in Arabidopsis, alfalfa, avocado, rice, soybean, and tomato.^{179,186,189,190} One important factor that may cause variations in transgene expression within different organisms is the complexity of host-plant system. This is because most of the experiments have been performed over the model organisms.

5. Greenhouse Vs. Field Performance of Transgenics

After successful transgene integration, transgenics are shifted to greenhouses where the initial experiments are carried out for evaluation of transformed genes against stress under study. Many experiments confirm that transgenics perform variably in different controlled conditions. According to a study carried out by Yao et al.,¹⁹¹ the transgenic events of sugarcane varieties B2, B36, B38, B48, and B51 were analyzed under both greenhouse and field conditions. The obtained outcomes of transgenics were also compared to the wild type varieties. The expression of integrated transgene Coat Protein of the SCMV (Sugarcane Mosaic Virus) was found similar in all the varieties but all of them performed differently under different environmental conditions. B2, B36, B38, and B51 were found highly resistant in greenhouse conditions against inoculated SCMV while B48 responded as entirely immune. Similarly, under field conditions, B2, B38, and B51 responded as moderately resistant while B36 responded as resistant and B48 responded as highly resistant as compared to the wild type which was highly susceptible to SCMV at all times. In another study, a wheat genotype, Line-32A, was transformed with thaumatin-like protein (tlp) gene from the O. sativa. Moderate resistance against the Fusarium graminearum was induced in 32A by expression of this gene under greenhouse conditions. Transgenic line was later grown under field trials where significant reduction in disease resistance was observed.¹⁷⁸ Baxter et al.¹⁹² reported the variable performance of switchgrass exhibiting overexpressed miR156 gene under different environmental conditions. It was concluded that the yield and resistance to certain diseases of transgenics was found to have been increased in greenhouse conditions while in field, these transgenics behaved differently. Their yield was found to be even on a level as compared to the control group and they also got highly susceptible to various diseases including rust. In another study, potato was transformed with RB gene against Phytophthora infestans to get resistance against late blight disease. The developed transgenic potato was tested under both, greenhouse and field conditions. It was concluded that strong foliar resistance was expressed by transgenics grown in the greenhouse. As they were moved from the greenhouse to field, the reduction in disease resistance was observed which eventually reduced and plant became susceptible to the disease once again. The exact reason behind these variations is still unknown but it is believed that it could be due to complexities present in open environment to which few genes possibly cannot comply.¹⁹³

6. GM Crops of Pakistan

Cotton and maize are the two major GM crops in Pakistan that are developed with resistance properties against insects and weeds. In 2002, Bt cotton was developed for first the time as a genetically modified crop in Pakistan. In 2005, Pakistan atomic energy commission (PAEC) commercialized four varieties of Bt-cotton exhibiting insect-resistance (IR) i.e. IR-CIM-443, IR-CIM-448, IR-NIBGE-2, and IR-FH-901 across the Pakistan to sort out the issue of insect attack which was getting epidemic. These genotypes reduced pesticides use resulting in increased farm income.¹⁹⁴ In 2011–2012, Punjab Seeds Council (PSC) has approved a commercial release of up to 40 different IR Bt-cotton varieties which are listed in Table 5.¹⁹⁵ Between 2013 and 2016, 50 more Bt-cotton varieties were approved by the National Biosafety Committee (NBC), PSC, and the Pakistan Central Cotton Committee (PCCC) for commercialization.^195–197 At present, 96% of the total cotton production in Pakistan is Bt cotton which is planted on a total area of 3 million hectares.¹⁹⁸ Some of the reported Bt-cotton varieties along with their approval year and developer institution are listed in Table 5. These varieties are backcross of Mon-531 event which carries Cry1Ac gene from Monsanto. This gene was patented in the USA but not in Pakistan, therefore once it entered Pakistan through unknown sources, it was used to breed resistance against major chewing insects in local cotton varieties.¹⁹⁹ This event successfully controlled major chewing insects of cotton in Pakistan. However, in 2015 resistance breakage against Pink bollworm (Pectinophora gossypiella) was reported.²⁰⁰ This resistance breakage was affiliated with the mismanagement of Bt cotton. Precautionary measures were adopted through governmental control in next three seasons which resulted in the recovery after the reported breakage.

Table 5.

Commercialized local varieties of Bt cotton in Pakistan.

Year of Approval	Variety	Developer
2002	FH-901	National Institute for Biotechnology and Genetic Engineering, Faisalabad
	CIM-443	National Institute for Biotechnology and Genetic Engineering, Faisalabad
	CIM-448	National Institute for Biotechnology and Genetic Engineering, Faisalabad
2006	NIBGE-2	National Institute for Biotechnology and Genetic Engineering, Faisalabad
2009	CEMB-2 (Hybrid)	Center of Excellence in Molecular Biology, University of Punjab, Lahore
2010	NIBGE-1524	National Institute for Biotechnology and Genetic Engineering, Faisalabad
	GM-2085 Hybrid	M/s Guard Agricultural Research Services, Lahore
	NIBGE-901	National Institute for Biotechnology and Genetic Engineering, Faisalabad
	NIBGE-3701	National Institute for Biotechnology and Genetic Engineering, Faisalabad
	FH-113	Cotton Research Institute, Ayub Agricultural Research Institute, Faisalabad
2011	Ali Akbar-703	M/s Ali Akbar Seeds, Multan
	Ali-Akbar-802	M/s Ali Akbar Seeds, Multan
	IR-3701	National Institute for Biotechnology and Genetic Engineering, Faisalabad
	MG-6	M/s Nawab Gurmani Foundation
	Neelum-121	M/s Neelum Seeds, Multan
	Sitara-008	M/s Nawab Gurmani Foundation
	NIBGE-4 (Discontinued in 2016)	National Institute for Biotechnology and Genetic Engineering, Faisalabad
2012	NS-141	M/s Neelum Seeds, Multan
	Sitara-10M	Aziz Group, Pakistan
	AA-904	Ali Akbar Seeds, Lahore
	Sun-1	Suncorp Pesticides, Multan
	Sitara-12	Aziz Group, Pakistan
	Auriga-213	Auriga Seed, Lahore
	KZ-389	Kanzo Quality Seeds, Lahore
	MNH-886 (Discontinued in 2016)	Cotton Research Station, Multan
	TARZAN-1	Four Brothers Seeds Corporation Pakistan Pvt. Ltd.
	AGC-777	M/s Weal AG Corporation, Multan
	BH-178	Central Cotton Research Institute, Multan
	AA-919	Ali Akbar Seeds, Lahore
	BS-52	HI SELL Seed Industry, Multan
	CEMB-33	Center of Excellence in Molecular Biology, University of Punjab, Lahore
	CIM-598	Central Cotton Research Institute (CCRI), Multan
	FH-114	Cotton Research Institute, Ayub Agricultural Research Institute, Faisalabad
	NS-161	M/s Neelum Seeds, Multan
	Leader-1	Suncorp Pesticides, Multan
	NIBGE-3	Nuclear Institute for Biotechnology and Genetic Engineering, Faisalabad
	Silkee	HI SELL Seed Industry, Multan
	VH-282	Cotton Research Station, Vehari
	NIAB-1	Nuclear Institute of Agriculture and Biology, Faisalabad
	GH-142	Central Cotton Research Institute, Multan
	CIM-591	Central Cotton Research Institute, Multan
	NIA-80	Nuclear Institute for Agriculture, Tandojam
	CRIS-510	Central Cotton Research Institute, Sakrand
	VH-300	Cotton Research Station, Vehari
	IUB-11	Islamia University, Bahawalpur
	CIM-612	Central Cotton Research Institute, Multan
	CEMB-44	Center of Excellence in Molecular Biology, University of Punjab, Lahore
	BH-180	Cotton Research Station, Bahawalpur
	CRIS-508	Central Cotton Research Institute, Sakrand
	BZU-75	Bahauddin Zakariya University, Multan
	VH-303	Cotton Research Station, Vehari
	MNH-456	Cotton Research Station, Multan
2013	A-555	Ali Akbar Seeds, Lahore
	FH-142 (Discontinued in 2016)	Cotton Research Institute, Faisalabad
	IUB-222	Agri. Farm Service, Multan
	KZ-181	Kanzo Quality Seeds, Lahore
	NIAB-824	Nuclear Institute of Agriculture and Biology, Faisalabad
	TARZAN-2	Four Brothers Seeds Corporation Pakistan Pvt. Ltd.
	SAYBAN-201	Kanzo Quality Seeds, Lahore
	Sitara-009	Sitara Seed Company
	Sitara-11M	Aziz Group, Pakistan
	TARZAN-3	Four Brothers Seeds Corporation Pakistan Pvt. Ltd.
	AGC-999	M/s Weal AG Corporation, Multan
	BH-184	Cotton Research Institute, Ayub Agricultural Research Institute, Faisalabad
	CA-12	M/s Ali Akbar Seeds, Lahore
	CIM-595	Central Cotton Research Institute, Multan
	CIM-599	Central Cotton Research Institute, Multan
	CIM-602	Central Cotton Research Institute, Multan
	FH-Lalazar (Discontinued in 2016)	Cotton Research Institute, Ayub Agricultural Research Institute, Faisalabad
	IUB-13	Islamia University, Bahawalpur
	Leader-3	Suncorp Pesticides, Multan
	MNH-988	Cotton Research Station, Multan
	NN-3	Nuclear Institute for Biotechnology and Genetic Engineering, Faisalabad
	NIBGE-5	National Institute for Biotechnology and Genetic Engineering, Faisalabad
	NIBGE-6	National Institute for Biotechnology and Genetic Engineering, Faisalabad
	SAYBAN-202	Auriga Seed Corporation, Lahore
	VH-259	Central Cotton Research Institute, Multan
	VH-305	Cotton Research Institute, Ayub Agricultural Research Institute, Faisalabad
	CEMB-66 (Discontinued in 2016)	Center of Excellence in Molecular Biology, University of Punjab, Lahore
	CA-926	M/s Ali Akbar Seeds, Lahore
	Sitara-13	Sitara Seed Company
	Leader-5	Suncorp Pesticides, Multan
	RCA-333	Nuclear Institute of Agricultural Biology, Faisalabad
	TARZAN-4	Four Brothers Seeds Corporation Pakistan Pvt. Ltd.
	CIM-616	Central Cotton Research Institute, Multan
	Cyto-177	Central Cotton Research Institute, Multan
	CMEB-55	Center of Excellence in Molecular Biology, University of Punjab, Lahore
2014	CEMB-77	Center of Excellence in Molecular Biology, University of Punjab, Lahore
	CIM-622	Central Cotton Research Institute, Multan
	Cyto-178	Central Cotton Research Institute, Multan
	NIBGE-7	Nuclear Institute for Biotechnology and Genetic Engineering, Faisalabad
	VH-327	Central Cotton Research Institute, Multan
	NIAB-874B	Nuclear Institute of Agricultural Biology, Faisalabad
	IUB-63	Islamia University, Bahawalpur
	Cyto-177	Central Cotton Research Institute, Multan
	BS-70	HI SELL Seed Industry, Multan
	AGC-Nazeer-I	M/s Weal AG Corporation, Multan
	Sitara-14	Sitara Seed Company
	Auriga-215	Auriga Seed, Lahore
2015	CIM-600	Central Cotton Research Institute, Multan
2016	FH-118	College of Agri. & Environmental Sciences, Islamia University, Bahawalpur
	MM-58	Islamia University, Bahawalpur
2017	Cyto-179	Central Cotton Research Institute, Multan
	FH-326	Cotton Research Station, Faisalabad
	NIAB-878B	Nuclear Institute of Agricultural Biology, Faisalabad
	BPC-11	M/s Biocentury Seeds, Lahore
	BS-15	M/s Bandesha Seed Co, Jahanian
	Koonj	ARI, Tandojam
	Sahara-120	M/s Patron Seed Company
	Sahara-150	M/s Patron Seed Company
	SHAHKAR	M/s Weal AG Corporation, Allah Din Group of Companies, Multan

Source: NBC, PCCC, PSC

Besides the insect attack, maize crop in Pakistan also grows at the verge of weed stress. Such issues have been claimed to be resolved by introducing and commercializing various GM/Biotech herbicide tolerant (HT) and insect resistant (IR) varieties of maize, including some hybrids harboring both traits (HT+IR) (Table 6). These biotech varieties were approved by PSC and NBC in 2016 and their cultivation started in early 2017 in Punjab and KPK province. Initial results show a remarkable increase in yield. With the adoption of these IR/HT maize varieties, the net profit of $1 billion (USD) to the farmers has been estimated in the upcoming decade.^201–203

Table 6.

Approved varieties of Bt maize in Pakistan.

Year of Approval	Variety	Developer
2017	NK603	Monsanto, Pakistan
	MON89034 × NK603	Monsanto, Pakistan
	TC1507× NK603	Duponut Pioneer, Pakistan
	TC1507× MON810× NK603	Duponut Pioneer, Pakistan

Source: ISAAA

7. Genetic Transformation Scenario in Pakistan

Many biotech research institutes have been working in Pakistan for over two decades, several transformation approaches have been developed in crops like wheat, cotton, and Brassica under local conditions for the delivery of desired genes into the target plant genomes. These approaches include floral-dip method, floral spray, biolistic, electroporation, vacuum infiltration, PEG-mediated, Agrobacterium-mediated transformation, and CRISPR/Cas system.^60,204Agrobacterium-mediated transformation is the most common, simple, and effective strategy used from the list of all strategies applied in both monocots and dicots. Most of these techniques have been used in the last two decades but CRISPR remains to be the most rapidly emerging and successful applied transformation technology at the moment. CRISPR/Cas9 plays dual role in activation and silencing of a specific gene, as it can induce site-directed mutagenesis or alteration within regulatory genes, encoding transcriptional activator or repressor proteins. With the help of this technology, functional genomics, genetics, epigenetics, and molecular biology of a gene/organism can be studied thoroughly.^204,205 The system can easily be reprogrammed by changing sequence of guide RNA as per desired gene.

The transformants obtained by implementing this approach exhibit more than one gene copy and show either transient or stable gene expression which varies from case to case. The high frequency of transformants and their inheritance in upcoming generations have been reported by studying the gene transmission over generations.²⁰⁶ The CRISPR-based transformants ensure biosafety and offer much help in combating major disasters like climate change and yield loss in agriculture sector. The gene flow to other native species can cause improvement in those lines as well. Though there can be a possible loss of natural resources, this approach can still help fulfill challenging requirements of stress scenarios. As this approach is entirely based upon synthetic molecules i.e. sgRNA, there would be no hurdles like bioethical issues. The role of this gene disruption technology is promising to the human health as well. It is the breakthrough of twenty-first century as it offers great solutions in addressing major challenges of time. The commercialization of such GMOs and their products can be perceived as the future which cannot be neglected.

8. Approved GM Events across the Globe

According to International Service for the Acquisition of Agri-Biotech Applications (ISAAA), the total number of approved and commercialized GM events reported all over the world is 508. That includes 30 different plant species which have been modified for the sake of improvement in their growth, quality, yield, biotic, and abiotic stress tolerance. Most of the events reported are hybrids as they exhibit two or three improved traits at a time. Tables 7 and 8 below list worldwide-reported GM events based on the plant species and improved traits by 2018.²⁰⁷

Table 7.

Global GM events reported in plants.

Events	Plant Species	No. of Events Reported
GM Events on basis of Plants	Medicago sativa Malus × Domestica Brassica napus Phaseolus vulgaris Dianthus caryophyllus Cichorium intybus Gossypium hirsutum Agrostis stolonifera Solanum melongena Eucalyptus sp. Linum usitatissimum  Zea mays  Cucumis melo Carica papaya Petunia hybrida Prunus domestica Brassica rapa Populus sp. Solanum tuberosum  Oryza sativa  Rosa hybrida Carthamus tinctorius  Glycine max  Cucurbita pepo Beta vulgaris Saccharum sp. Capsicum annuum Nicotiana tabacum  Lycopersicon esculentum Triticum aestivum	5 3 41 1 19 3 63 1 1 1 1 231 2 4 1 1 4 2 48 8 2 2 40 2 3 4 1 2 11 1

Source: ISAAA

Table 8.

Global GM events reported regarding traits.

Events	Improved Traits	No. of Reported Events
GM Events on basis of Traits	Abiotic Stress Tolerance Altered Growth/Yield Disease Resistance Herbicide Tolerance Insect Resistance Modified Product Quality Pollination Control System	11 3 28 342 294 92 31

Source: ISAAA

9. Approved GM Events in Pakistan

According to the GM Approval Database of ISAAA (2018), Pakistan exhibit only 6 GM events that are approved for cultivation and commercialization. Two of them are reported in cotton for insect resistance while other four in maize for herbicide tolerance or hybrids having insect resistance approved between 2010 and 2017. The genes involved in these events are Cry1Ac, Cry1Ab-Ac, Cry1Fa2, and CP4 Epsps that were taken from bacterial sources.

10. Funding of Transformation Project by Major Agencies in Recent Years

The research on GMOs started almost two decades ago in Pakistan. This was the time when HEC took off and started funding research and development projects. Apart from HEC some other funding agencies like Punjab Agriculture Research Board (PARB), Agricultural Linkages Program (ALP) controlled by the Pakistan Agricultural Research Council (PARC), and Pakistan Science Foundation (PSF) have started funding various projects. The United States Agency for International Development (USAID), International Center of Agricultural Research in Dry Areas (ICARDA), International Food Policy Research Institute (IFRI), Center for Environmental Risk Assessment (CERA) have also funded various projects in Pakistan. We tried to explore national funding agencies to get an awareness on how many projects they have funded; however, we could only get this information from online websites of PARB, ALP, and PSF. It was not possible for us to get information from HEC, through their website or by means of sending them repeated e-mails. HEC is the major funding agency which awards funding of the research projects in the universities and/or degree-awarding institutions.^208–210

If we compare Table 9 with Tables 7 and 8, it becomes evident that Pakistan is not following the global trend. Cotton, maize, and soybean are three major crops and herbicide tolerance, insect tolerance, and quality characters are three major traits incorporated in these crops. Table 9 shows that PSF has funded 9 projects and all of them on different crop plants; PARB has funded 10 projects on 7 different crop plants while ALP has funded 8 projects on 5 different crops; woody and fruit trees are altogether absent from this list. In order to understand the fact that priority should be given to crops based on their status in country’s agricultural economy and nutritional requirements, it does not seem to exist when we see the list of awarded projects by these funding agencies in Pakistan. Here, it is worth mentioning that rice is an export commodity of Pakistan and its transformation has been discouraged in recent years and there remains no possibility of field permission for GM rice in Pakistan. Bt-eggplant was developed by transforming Cry1Ac into it to combat the eggplant fruit and stem borer which is a major issue in Bangladesh and the Philippines. Therefore, with the collaboration of these two countries Bt-eggplant was established in 2003 and after seven consecutive years of greenhouse trials the step toward field trials was taken right after assuring biosafety and assessing possible risks. Remarkable increase in the yield of crop was observed within greenhouse and in field. In 2010, the approval for cultivation of Bt-eggplant was given for the first time to some limited areas and in 2016 Bt-eggplant was commercialized.²¹¹ Pakistan should also look for such international ventures and should find its partners in the public sector. This is because partnering with private companies usually involves royalties leading to expensive technologies for poor farmers while the cheapest technologies can be disseminated to the poor when public sectors work together.

Table 9.

Funded research projects on GM crops by three major agencies in Pakistan.

Agency	Year/Status	Research Projects
ALP	2002	Investigation of Role of Germin-like Proteins (GLPS) During Germination/Early Development by Construction of Rice Plants Engineered for Sense and Anti-sense Expression of Rice GLP
	2004	Transgenic Tomato with Resistance to Bacterial Wilt
	2005	Use of RNA Interference for Genetically-Engineered Male Sterile Tomato Plants for Production of Hybrid Tomato
	2008	Development of Salt Tolerance in Sugarcane through Genetic Engineering
	2014	Enhancing Fertilizer Use Efficiency in Wheat by Using Transgenic Approach
	2018	Development of Bio fortified Tomato with Precursor of Vitamin A
		Utilization of Multiple Transcription Factor Genes for Enhancing Wheat Yield
		Genetic Transformation of Chickpea for Herbicide Resistance
PARB	Completed	Genetic Improvement of Groundnut for Herbicide and Disease Resistance
		Transgenic approach to improve drought and salinity-tolerance in wheat
		Introgression of cotton leaf curl virus resistance genes from Gossypium arboreum (Desi Cotton) into Gossypium hirsutum (Upland Cotton)
	Ongoing	Genetic engineering of fiber trait genes to improve staple length in cotton.
		Engineering Potato Against Frost and Viruses
		Expression of cell-wall degrading enzymes in higher plant chloroplasts for bioethanol production from lingo-cellulosic plant biomass
	Ongoing	Genetic Improvement of cotton for herbicide and bollworm tolerance
		Genetic improvement of Sugarcane for Herbicide and Borer Resistance
		Development and commercialization of indigenous BT and herbicide tolerant maize hybrids
		Development of transgenic cotton with multiple genes resistant to cotton leaf curl virus
PSF	Completed	Engineering Phage-type chimeric promoters to over express Bi-functional proteins in rice protoplastids to develop biosafe transgenic plants.
		Increasing Phosphorus use Efficiency in Wheat Through Genetic Engineering.
		Production of Genetically Engineered Oilseed rape for Enhanced disease Resistance
	Ongoing	Genetic Engineering of Sugarcane with the Rice Tonoplast H+ PPase Gene to Improve Sucrose Content and Salt Tolerance
		Genetic Transformation of Brassica Carinata for Low Viscosity Biodiesel Production
		In Vivo Evaluation of Anti Sense Poly Phenol Oxidase Gene Construct Under the Control of a Wound Inducible Promoter
		Engineering Maize with Heat Shock Proteins.
		Development of Homozygous Lines of Transgenic Wheat and Screening for Phosphorus Use Efficiency
		Development of Cost Effective and Potential Biocontrol Agents for Area Wide Management of Sucking Pests in Bt Cotton

Source: ALP, PARB and PSF websites

11. Socio-Economic Impact

The developing countries like Pakistan must maintain a balance between bioethics, environmental concerns, and sustainable food security. Around 24.3% (~51 million people) of the total population of Pakistan which accounts to 210 million people live in poverty.²¹² There is prevalence of disparities among people based upon their lifestyle, gender, and region. Agriculture has the largest share of GDP of Pakistan, and many industries depend on it for their supply of the raw materials.²¹³ Conventional plant breeding techniques have successfully fulfilled the seed needs of the country and have increased yield at excellent pace. Pakistan is among the top 10 countries of the world on list of yielding capacity for multiple crops. Recombinant DNA technology has contributed significantly globally in improvements in health, livestock and agriculture sectors. Social consensus on GMOs acceptability is still too far from being achieved while the adoption of genetic engineering techniques for exploring and interpreting complex biochemical processes, development of the new medicinal drugs, and comprehensive understanding of disease mechanisms is remarkable.^214–217

In recent action plans, government of Pakistan claims biotechnology as a superior area of research which exhibits great potential for increasing crop and livestock production, and large proportion of country’s economy depends upon agriculture and livestock. The research projects worth $16.7 million are being funded by the government to encourage progress in the field of biotechnology and genetic engineering. Though Pakistan is among the countries that invest least amounts of money on their research and development sector, still many publications and patents are being produced. Relevant research is being progressed in only 50 HEC-recognized biotechnology research institutes across the country.⁷ The research projects are ongoing for the development of virus-free, herbicide-tolerant, disease-resistant, salt-tolerant, and drought-tolerant genotypes of wheat, cotton, canola, sugarcane, potato, and tomato. In addition, the development of hybrid seed by male sterility is also being produced.²¹⁸ Public money invested into this kind of research is expected to bring positive change in the lives of the people. It will be worthwhile to dedicate more funding to the top priority areas that have demonstrated success in the world instead of distributing money haphazard. This has become even a more delicate issue when a 50% budget cut for HEC in FY-2019-20 is done.²¹⁹

12. Establishment of National Biosafety Committee

American biosafety rules for the regulation of genetically modified crops were introduced in 1984 while similar rules in China were established in 1997 at the very beginning of the Biotechnology era. While the research on genetically modified crops and biosafety in Pakistan started in 2005 with the establishment of biosafety rules. These biosafety rules were passed to ensure product safety, trade of GMOs, and research and development regulations. Additionally, the guidelines for monitoring and evaluation systems were also developed to implement these rules.²²⁰ Though Pakistan has emerged in research in this field later than other countries still in spite of having limited relevant research technology and institutions, it was able to establish a framework of biotechnology research. Pakistan has established the National Biosafety Committee that has met approximately 20 times, and has already discussed and approved various cases initiated by the 35 institutional biosafety committees (IBCs) which have been founded so far for the purpose of supervising the ongoing research based on the Pakistan Biosafety Act 2005 Figure 3.

Figure 3.

Process of GM Approval in Pakistan.

Institutional Biosafety Committees, TAC (Technical Advisory Committee), and NBC are different tiers to be followed at the stages of initiation of research, field evaluation, and import of GM technology. Once an event is cleared by NBC, it is handed over to the provincial bodies like PSC, followed by the varietal approval process for commercializing in the country.²²¹ As an outcome of these accomplishments, Bt cotton genotypes are approved and cultivated for over a decade so far in Pakistan. Insect-resistant and herbicide-tolerant maize genotypes that were approved for general cultivation since 2017 can be seen in Table 10.

Table 10.

GM events reported in Pakistan.

Event	Crop	Year of Approval	Trait	Gene
MON531	Cotton	2010	Insect Resistance	Cry1Ac
GFM Cry1A	Cotton	2012	Insect Resistance	Cry1Ab-Ac
NK603	Maize	2017	Herbicide Tolerance	CP4 EPSPS
MON89034 × NK603	Maize	2017	Herbicide Tolerance Insect Resistance	CP4 EPSPS
NK603 × MON810	Maize	2017	Herbicide Tolerance Insect Resistance	Cry1Ab CP4 EPSPS
TC1507 × NK603	Maize	2017	Herbicide Tolerance Insect Resistance	Cry1Fa2 CP4 EPSPS

Source: ISAAA

13. Science and Politics in Pakistan’s GMO Regulation

Since its birth, NBC had been working on regulating research and commercialization of genetically modified crops. There are strict biosafety rules enforced for the monitoring and evaluation and transporting of GMOs under the supervision of a designated Directorate established under NBC by the Ministry of the Environment. It also worked on the risk assessment and labeling of imported GMOs that are openly available in the markets without any tags or labeling.²²² After a thorough inspection, Biosafety Committee holds the right to ban the import/export and sale/purchase of any product which may pose any threats to the human health or the environment.

The acceptance of GM crops by farmers in the very beginning was simply an easy task as the management cost of GM crops was comparatively less than that of non-GM ones which ultimately attracted the farmers and led to commercialization of GM varieties. The same happened with Bt cotton introduction to Pakistan. Another major reason behind this acceptance was dearth of relevant research and knowledge of controversies. It is reported that almost all cotton-occupied land in Pakistan was cultivated with Bt-cotton within two years from its introduction in 2005.^223–225 Seed Act (1976) did not accommodate for the registration, regulation, and certification of GM seed. In 2015, the amendment in the Seed Act was made based on the research which helped in opening the door for commercialization of GM crops,²²⁶ though varietal approval process came in place well before that as stated previously in Section 12.

As soon as GMOs made their way into the Pakistan agriculture, different lobby groups became active, which started a debate at many public forums including the Parliament. The Parliament has started a discussion about banning GM maize in Pakistan because of the rumors that it had carcinogenic compounds in it referring to some reported unsubstantiated cases across the globe. Representatives of these lobby groups also claimed that the research facilities of Pakistan are not capable of handling and monitoring the regulation of such GMOs.^227,228 National Biosafety Committee was established under EPA which was part of the Ministry of the Environment. After the 18^th Amendment was passed, environmental issues became a subject of provincial-level discussions. Earlier on, biosafety rules were approved under Cartagena protocols to which federal government was signatory. Legal battle has started over the future of GMO crops in Pakistan but as of now its process has been halted. Many experiments are yet to get approval and many products are shelved due to this legal battle. In 2014, an order was issued by the Lahore High Court to the Federal Government of Pakistan for the issuance of licenses to the producers of GM cotton and maize which was later suspended within a month upon the appeal filed by the Federal Government.²²⁹ The Ministry of Climate Change of Pakistan has clearly restricted commercialization of GM crops without thorough investigation of research practices and regulations of GMOs presented by Biosafety Act 2005. Pakistan Environment Protection Agency (Pak-EPA) and NBC still demonstrate optimism regarding commercialization of GM crops. Various GM varieties have been approved in 2016 while other crops are still under ongoing research trials.^227,230 GM corn is approved in Pakistan but still the debate has halted its way to farmers’ fields.

14. Risk Assessment

For the commercialization of GMOs, risk assessment is a matter of a prime importance as it focuses on the effects of the product upon non-targeted organisms and overall biodiversity.^60,231 The systems of risk assessment and management are usually established upon the designed framework for the living modified organisms (LMOs) in accordance with the Cartagena Protocol on Biosafety (CPB) and Convention on Biological Diversity (CBD).⁶ These protocols were taken as guidelines for formulation of Pakistani biosafety rules in 2005, hence these rules stress the need of a thorough risk assessment studies before approval of GM crops. At the same time, the performance of the claimed transgene is also considered in terms of its expression and efficiency. PSC rejected 19 Bt-cotton varieties including FH-142, FH-Lalazar, and MNH-886 due to their poor performance including susceptibility to various diseases and failure to deliver better yield results which were promised at the stage of their approval. In order to improve the risk assessment process, Center of Environmental Risk Assessment (CERA), a Washington-based agency launched a funding program from 2011 to 2014 under South Asia Biosafety programs and funded more than 20 projects. Interesting results were shown from these projects as they were presented in second South Asia Biosafety Conference (Annonymous, 2014. 2nd South Asia Biosafety Conference https://ilsirf.org/event/2nd-annual-south-asia-biosafety-conference/) held in Colombo, Sri Lanka, on September 2014.^212,232

After developing a transgenic event, its biosafety has to be confirmed prior to approval process which can be done by Real Life Risk Simulation (RLRS). It involves a cascade of trials i.e. in vitro and in vivo in order to any sort of ambiguity that could possibly occur. However, the obtained findings are then cross-checked by supervising approval organizations. RLRS is another phenomenon of using an alternative model organism to get some tasks executed by following recommended bioethics and biosafety rules and regulations. It is being used for several years to ensure biosecurity of existing wild-type species from getting toxic by evaluating their toxicity tolerance threshold^233–236 and similarly, with the exposure of some novel and modified organisms, biosecurity of biodiversity have to be ensured. The imitations are performed for the testing of transgenics through field and open trials by the approval committee for commercialization of these transgenic events.^237–239

15. Conclusion

Pakistani farmers are among the major Pro-GMO farmers in the world who have been cultivating GM cotton for over a decade. The adoption of Bt cotton by small farm holders has caused affirmative impacts on the dietary quality and food security. It is evident that GM crops are a significant component of the food security. Besides, GM crops also act as a panacea for malnutrition and hunger. The main focus of the research institutions in Pakistan had been bound to the development of insect-resistant and herbicide-tolerant crops in the past. Research is also being done on different crops in order to combat the major challenges of global warming and climate change. Though the success rate of GM varieties against abiotic stress factors is lower than that of the biotic ones still the researchers are trying hard to develop such material. Research on transgenics has found that the reason behind success of HT/IR varieties is the selection of their genes (ALS, AHAS, Bar, Cry1Ac, Cry2Ab) from unique and distinctive sources such as bacteria which do not interfere in the cellular mechanisms of the plant. In case of abiotic stress tolerance, selection of resistant/tolerant genes is done from the same living system but different species. The genes products tend to act upon off-targets sites/mechanisms which ultimately causes hurdles in obtaining desired trait. Researchers should focus on the gene sources which do not interfere with the internal plant system and give maximum responses to stress. Government of Pakistan is financing almost all hi-tech research institutions for the purposes of carrying out research projects that focus on the discovery of appropriate ways to tackle the disastrous effects of the climate change.

Funding Statement

The lab of the principle author from Pakistan is supported by the Pakistan Science Foundation [NSLP/(AU)168].

Highlights

• Various stress factors imposed by the climate change significantly reduce the yield per unit area in Pakistan.

• Plethora of basic and applied gene transformation studies show the potential of developing GM crops with sustainable yields and quality under stressed environment

• Regulatory protocols are in place for risk assessment and approval of GMOs in Pakistan.

• GMOs are very much accepted at famers’ field and breeding stations in Pakistan but policies of the local and multinational companies have been hindering the access to many GMOs cultivated worldwide.