Conventional Breeding, Molecular Breeding and Speed Breeding; Brave Approaches to Revamp the Production of Cereal Crops

Conventional plant breeding methods exploit already existing genomic variation in plants to develop a variety in 8 to 10 years, which can decrease the genetic variability of plant’s genome. The ever-increasing food demand of cereals crops cannot be met by the traditional breeding methods. In order to increase the food production in less time, there is a dire need to improve the breeding methods. Several conventional and molecular breeding methods are being used to improve the crops traits. Molecular researchers have developed new genome editing tools like CRISPR/Cas9, CRISPR/Cpf1, prime editing, base editing, dcas9 epigenetic modification, and several other transgene free genomes editing approaches. These genome editing tools can improve the desired traits precisely and efficiently. Moreover, a newly developed breeding method “Speed Breeding” has revolutionized the agriculture by shortening the crop cycle. It can produce 5-6 generations of cereals in a year. In this review, we have summarized all these conventional and molecular breeding approaches to improve the cereal crops.

Meganucleases were the first genome editing tools to improve the maize and wheat genome.
Meganucleases are naturally occurring molecular DNA scissors that can recognize up to  DNA bases. Zinc finger nucleases work on the same pattern as meganucleases and can recognize up to 9-18 base pairs. TALENs have advantage over other site-specific nucleases (Meganucleases and Zinc Finger nucleases) due to its nature that it targets at single nucleotide as compared to three in meganucleases and zinc finger nucleases (ZFN). TALENs were successfully used in many cereal crops 6 . CRISPR/Cas9 and CRISPR/Cpf1 were associated with off target mutations, and this issue was resolved by using the modified genome editing tools such as prime editing, base editing and dCas9 based epigenetic modification 7 . Apart from molecular approaches, an advanced breeding method called "Speed Breeding" was introduced in simple breeding procedure 8 . Speed breeding speeds up the breeding methodologies with short generation period and have refined to achieve up to 6 generations in one year 9 .
In this review, first we discussed about the role of conventional breeding approaches, and then compared it with the new genome editing tools and speed breeding approach for the improvement of cereals. Finally, we have discussed about the applications of these approaches to increase the cereals production. In result of GE, two kinds of repairing mechanisms are switched to edit the plant genome (NHEJ and HDR). NHEJ is a non-homology repairing mechanism which can be substituted by insertion or deletion of specific part of genome, naturally. Left is HDR repairing mechanism, can be edited according to our choice, and insert donor DNA

Mutation Breeding (Conventional Breeding) Mechanism and Its Role in Plant Breeding:
Plant breeding is a technique that is used for the development of superior plants. Its performance depends upon the availability of genetic variations. By making crosses, genetic variability is exploited to transfer it in new varieties 10 . Many other conventional breeding approaches like mutation breeding is also used for the development of new cereal crop varieties 11 .
Mutation causing agents called mutagens are categorized in two categories, namely physical mutagens and chemical mutagens 12 ' 13 . In contrast of physical mutagens, chemicals mutagens are solely used for the point mutation. Physical mutagens ( Table 1) is comprised of ionizing radiations which can alter the genetic makeup 14 .  Chemical mutagens (Table 2) are also important for creating point variation in plants genome, but its effects are milder than physical mutegens 9 . The exposure to mutagens causes DNA double strand breaks in plants. Plants have a mechanism to heal the broken strands which can lead to crop improvement. Basically, this phenomenon is the base of improving crops by mutation breeding 15 .
Past Achievements of Conventional Breeding in the Improvement of Cereals: In cereals many varieties were developed by using the mutation breeding ( Fig 5). Cereals are playing their vital role to meet the production needs. Though cereal production is more than other crops however, it is still difficult to meet the production target by 2050 with the continuously increasing population.
Historically, plant breeders were solely using introduction, selection, and hybridization technique to improve the cereals. But, in the present era, mutation breeding is only the solution to create genetic variation instantly in available germplasm. Up to 2020, many varieties by mutation were released by different countries across the globe ( Figure 6). A high yielding wheat variety "Stadler" developed by USA had also showed a dramatic resistance against leaf rust, loose smut, lodging, and also depicted excellent results for early maturity 17 . In Pakistan, the widely cultivated wheat varieties named as Jauhar-78, In the below diagram we have made a comparison between different GE tools (Table 3).
Recently, a wide variety of molecular approaches have surfaced.    . Two monomers bind at a specific genome region to work as a endonuclease and cut that part. (b) ZFN which also work as restriction enzyme. ZFN DNA-binding domains (module) contains Fok1 nuclease domain which bind in such a manner, cut the genome part between the binding sites. (C) TALENs are consisted of DNA binding and cleavage domains. DNA binding domain is composed of 33-35 repeated amino acids, with divergent 12th and 13th amino acids. These two variable positions are called as repeat variable di-residues (RVDs). Due to variable in nature, slight change in RVDs can change the targeting efficiency. These RVDs bind at specific region of the genome, along with nuclease domain (Fok1) that cut the specific genome part. In the given diagram, there is a complete procedure to edit the genome by GR tool like (a, b, c).

ZFN Mechanism:
ZFNs were discovered in 1980s which were the most versatile tool to edit the genome at users defined locations for the improvement of important triats 35 52 .

TALENS Mechanism:
Transcription activator-like effector nucleases (TALENS) were developed to edit the plant's genome more efficiently as compared to ZFNs. Journal "Nature method" enlisted TALENS as a method of the year due to its astounding performance for precise genome editing.

Developmental history of TALENs, is incomplete without intervention of phytopathogenic
Xanthomonas bacteria 53 56 . In contrast to ZFNs, TALEs are comprised of tandem repeats of 33-35 amino acids, and each repeat targets only single nucleotide which make it more flexible and precise genome editing tool. Each amino acid repeat is comprised of (RVDs) at positions 12 and 13. In 2009, for the first time, these RVDs were confirmed by Bonas and another group of researchers. Based on RVDs, each amino acids repeat is dictated to specify the single base pair and bind on targeted region 57 . These RVDs are highly variable in their nature so can bind at more than one targeted sites 58 . In pursuance of highly efficient and precise genome editing, these TALE repeats can be engineered to direct the binding of amino acids at specified DNA sequences 58 .  CRISPR from yogurt to Plant Breeding: It took more than a decade to understand the mechanism of Cas9 and its function as endonuclease to edit the genome which was thought to be due to the mysterious repetitive sequences, later named as CRISPR (clustered regularly interspaced short palindromic repeats) ( Figure 8). CRISPR loci is composed of Cas genes, repetitive sequences interspaced by variable sequences (spacers) which are corresponding to the sequences present in foreign genetic elements called as protospacers ( Figure 9). Cas9 genes translate themselves in proteins and degrade the genome of foreign genetic element. While, CRISPR array are transcribed into shorter CRISPR RNAs (crRNAs) but most of the CRISPR arrays are initially transcribed in a single RNA 73 . CRISPR was named for the repeated sequences and for the common associated genes present is clusters that were adjacent to the repeated sequences were labelled as "Cas" genes 74 . Further, it was observed that the viruses that infect bacteria shares some similarities with the sequences present between the repeats. The Cas genes were also identified having the ability to cut DNA by encoding domains of proteins 75 . These associated genes serves the basis of classifying CRISPR into 3 different types (I,II, III) 76 . The type I and III have different Cas proteins that also form complexes with CrRNA (CRISPR RNA) in order to assist the target nucleic acids identification and destruction 77 . The type II has a smaller number of Cas proteins and their biological importance is still elusive 78 . The type II led the basis of genome editing techniques. Figure 8: Schematic mechanism of bacterial CRISPR system as a defensive tool to degrade the viral genome. During the invasion (step 1: invasion of virus) foreign genetic material (virus) enters bacteria acquire spacers inside their genome and save it to recognize in future invasion and found between direct repeats. (step 2: spacer integration in CRISPR locus). CRISPR array is a noncoding part which is maturated and work only according to specific CRISPR system (step 3: CRISPR RNA formation and processing). In CRISPR type I and III, associated ribonucleases in CRISPR work to cleave the pre crRNA between the repeats and liberate many short crRNAs. System III associated crRNA further goes through a process at 3'end by employing the RNases which are yet to be identified and produce maturated RNA transcript. (Step 4: Destruction of target genome). For the recognition and destruction of the target sites, the type I and III has several complexes of proteins with crRNAs. The cascade complex is present in type I and Csm and Cmr complexes are present in type III for DNA and RNA cleavage, respectively. The cas3 nuclease bounded with the R-loop facilitates the process in type I. whereas, the type II has less proteins and cas9 is required for degradation. Protospacer adjacent motifs (PAMs) in type II facilitates the cas9 in identifying the target sites. In both I and II types, self-targeting of CRISPR is prevented due to the lack of PAM in the targeted sequences. CRISPR/Cas9 Mechanism: The development of CRISPR/cas9 mechanism (Fig 10) for the improvement of crops is associated with the bacterial defensive mechanism, as some bacteria and archaea put into service the CRISPR array to disrupt the viral genome eventually. CRISPR mechanism is performed in three steps: (1) Acquisition step: acquisition of spacer DNA from the viral DNA for which results in the insertion in bacterial genome (to memorize the invading viral DNA); (2) Expression Step: expression of CrRNAs from the transcription of CRISPR array which also involves the expression of the Cas9 protein; (3) Interference Step: CrRNA acts as a guide RNA which is further directed by Cas9 protein to bind at targeted DNA that is accompanied by PAM sites, and cut that specified DNA three nucleotide away from PAM sites at both DNA strands 79 .
In result of targeted DNA cleavage, DSBs occur that switches on the repairing mechanism of cell's machinery. Two kinds of mechanisms are switched on: (1)   After transformation in plant cell, following steps are carried out: activation of Cas9 proteins, cleavage at targeted sites, production of DSBs. Activation step-involves the gRNA to activate  84 . CRISPR/Cpf1 has been used in many plants 85 . Furthermore, it is necessary to insert or delete the nucleotide sequences for the improvement of crop traits.
For this purpose, naturally repairing mechanism of cell machinery is switched on. Generally, HDR and NHEJ nucleotide repairing mechanism works to insert the nucleotide sequences precisely at cleavage site or random insertion/deletions, respectively 86 .

Genome Editing without DSBs and Donor Template:
CRISPR-Cas9 is very versatile tool to edit the plant's genome precisely and with efficacy. His840Ala mutations containing dCas9 protein with other effector proteins to bind at specified genome location. It can alter the single base pair without any cleavage of that region 92 . This dCas9 protein has no more nuclease activity but work to guide the sgRNA for binding. Genome editing without DSBs. Generally, three kinds of GE approaches are being used to edit the plant genome without producing double stranded breaks; a, b, and c (Base editing, Epigenetic modification and Prime editing. In (a) by using the base editing approach, two genes (TaALS and TaACCase) were co-edited. This approach was used by coupling the dCas9 with Cytosine base editor (CBE). In this way such type of transgenic wheat plants were developed which did not produce any DSBs. (b) is Epigenetic editing. In this approach, dCas9-Suntag-hTET1cd was coupled with dCas9 for demethylation of FWA promoter to activate the FWA gene expression. (c) Prime editing, it works by developing a complex interaction between pegRNA, Cas9 nickase-reverse transcriptase (RT) and target DNA. In the pegRNA, except of primer binding site (PBS), desired genome sequence is also present which is introduced in the host genome. For RT, pegRNA produces primer. RT copies the information of pegRNA, and RT product is integrated in the target genome site. Initially, modification happens only at one target DNA strand. Later, it is present on both strand due to cell's repairing mechanism.
Base Editing: Precise genome editing requires gRNA, Cas9 protein, donor template, repairing mechanism for the editing of genome. While, base editing use the reprogrammable deaminase intending to introduce the bases at targeted site without any cleavage and induction of DSBs 93 . Nowadays, CBE (cytosine base editor) and ABE (adenine base editor) have been developed to alter the C-T and A-G, respectively 87 . In human, daily Spontaneous hydrolytic deamination causes conversion of C-T, A-G, 500 times per cell 94 . By doing the point mutations, diseases and specific traits can be improved. ABE contains different base editors, including Target-AID and BE. In Target-AID, pmCDA protein is fused with dCas9 protein (Cas9n, D10A) to perform base editing. In BE series rAPOBEC protein is used for fusing with dCas9 protein (Cas9n, D10A). CBE is used to alter the C-T, and then T is changed to U in response of natural repairing mechanism. CBE genome editing technique has been already used in crops: tomato, wheat, rice, maize and Arabidopsis. While, ABE is used to deaminase A to G, and reported in wheat, rice, Arabidopsis, and Brassica napus 87 .
Epigenetic Editing: Epigenetic refers to modification of genome without perturbing the DNA sequences such as histone modification, DNA methylation, DNA demethylation, gene imprinting, chromatin remodeling etc. 95 . These modifications are common in plants 96

Prime Editing:
Prime editing is also a new genome editing technique which utilizes the Cas9 nickase amalgamated with pegRNA to edit the genome precisely by "search and replace mechanism" 98 .
In CRISPR/Cas9 mechanism, DSBs are generated which are associated with some complex off target effects, including p53 activation, and translocations. 99    Genome Editing (Without DSBs) Role in Cereals Improvement: According to our understanding various groups used the base editing tool to improve the plants genome 193 .
First time a new DNA free gene editing tool (prime editing) was developed to make it workable for the mammals and yeast 98 . Later, this system was modified to develop the prime editor 2 However, this approach is time taking and laborious 198 .
Programmed Self-Elimination of transgene plants: In order to reduce the time and cost to select the transgenic free plants, in this approach two suicide genes are used for isolating the null segregants 200

RNP-mediated genome editing:
Genome editing using RNA is the most reliable method to be used for transgene free plants.
The RNA can be transferred to the protoplast through in vitro culture, as the RNA transformation is DNA-free, the transgene free plants can be obtained 206

Speed Breeding
Breeding by using conventional breeding methods have shown significant results for many years. Many varieties and lines have been developed by using conventional breeding methods.
But, with the passage of time, the dissemination of new improved cultivars is needed with substantial increase in production efficiency and disease resistance due to the overgrowing population 209

Achievements of Speed breeding:
In wheat multiple traits related to diseases like leaf rust and root architecture that were highly variable in both the field and in greenhouses required high throughput repeatable methods for screening was easily achieved by Speed breeding. It is cost efficient and took less time in screening as compared to the conventional breeding. This robust method allows to screen the germplasm more rapidly thus proved highly efficient for variable traits 131 . In wheat plant height, flowering time period and resistance to several diseases can be achieved by speed breeding 218 .
In Argentina, Scarlett is most extensively cultivated cultivar of barley which is susceptible to different diseases. By taking 4 lines with modified backcrossing methodology resistant lines were developed within 2 years that were disease resistant and yields more than the cultivar 210 .
In barley the glaucousness on the leaf sheath is an important trait for the plant to survive under hot climatic conditions 219 . This drought tolerant trait in Barley can be obtained by Speed breeding 218 ' 220 .
Rice is the most sensitive cereal crop to salt stress. Breeding to get salt tolerant varieties take many years which makes the task difficult. With the use of advance techniques like SNP and whole genome sequencing it becomes easier for the breeders to insert gene and then achieve several generations in each year with the help of speed breeding 221 . In rice a new salt tolerant line "YNU31-2-4" was developed with the help of speed breeding. After inserting genes by SNP, the breeding cycle was accelerated with the help of speed breeding methodology by using optimum light 14h light and 10h darkness from germination to 30th day to allow the plant to complete its vegetative phases and after it 10h light and 14 h darkness was provided to initiates the reproductive phase. The tillers were removed, and embryo rescue technique was used to save the time before seed maturity. Thus, enabling the researchers to get 4 to 5 generations of rice per year 129 .
A considerable improvement in breeding has been achieved as compared to DH technology which faces several agronomic drawbacks: low germination rate, poor vigor and sometimes distorted growth 222 . Moreover, developing DH lines are costlier as compared to speed breeding which don't require any specific precision 223 . The speed breeding with single seed descent (SSD) is useful to screen diverse germplasm within a short period of time by hastening the breeding cycles 8 . Single seed descent with speed breeding is time saving and cost efficient as compared to the conventional pedigree breeding method 211 . Moreover, speed breeding surpasses "shuttle breeding" and produces 3 times a greater number of generations. With shuttle breeding only 2 generations per year can be achieved while with speed breeding up to 6 generations can be obtained 224 .

Conclusion:
Two decades ago, conventional breeding was a major and easy approach toward improvement agronomic traits by exploiting the genetic variation that is caused by mutagens and cross breeding. In the present era, CRISPR/Cas9 is time efficient and a improved GE tool, as it inserts and deletes the DNA at specified genomic sites, either by NHEJ and HDR repairing mechanism. Despite of CRISPR/Cas9 importance, it is associated to limitations, including off target effects, and can cut only single targeted genomic site. Discovery of CRISPR/Cpf1 has played its multiplayer role by targeting the multiple genomic sites to improve the crops. Like CRISPR/Cas9, CRISPR/Cpf1 is also prone to off target effects which could lead to undesirable agronomic traits. Both GE tools require donor genomic part to put at the cleavage sites which is a complex procedure and could lead to undesirable effects in next generations.