WO2020063775A1

WO2020063775A1 - Method for knocking out bnmax1 gene in brassica napus l. using crispr-cas9 system and application

Info

Publication number: WO2020063775A1
Application number: PCT/CN2019/108240
Authority: WO
Inventors: 华玮; 郑明�; 张亮
Original assignee: 中国农业科学院油料作物研究所
Priority date: 2018-09-27
Filing date: 2019-09-26
Publication date: 2020-04-02
Also published as: AU2019345788A1; CN109266646A; CN109266646B

Abstract

Provided are a method for knocking out BnMAX1 gene in Brassica napus L. using CRISPR-Cas9 system and an application, two sgRNAs of specifically targeting Brassica napus L. BnMAX1 gene are designed and synthesized into oligo dimer, which are connected with Cas9 carrier and introduced into hypocotyl callus of Brassica napus L. by Agrobacterium-mediated genetic transformation technology to regenerate into shoots, Cas9 nuclease cleaves target sequence under the guidance of sgRNA, each sgRNA can mediate the cleavage of the BnMAX1 gene on the A and C genomes through the CRISPR-cas9 system, achieving the purpose of knocking out gene. Phenotypic identification shows that homozygous mutant lines increase the number of branches and pod number per plant, decrease plant height and increase yield.

Description

Method and application of using CRISPR-Cas9 system to knock out BnMAX1 gene of Brassica napus

Technical field

The invention belongs to the technical field of genetic engineering, and specifically relates to two sgRNAs specifically targeting the BnMAX1 gene of Brassica napus and a method for knocking out the BnMAX1 gene of Brassica napus by using a CRISPR-Cas9 system, which can be used for breeding lodging-resistant and high-yield rape varieties.

Background technique

Brassica napus (Brassica napus L.) belongs to Brassicaceae Brassica and is the second largest oil crop in the world in total production. It is also one of the most important oil crops in China. Its rapeseed oil accounts for edible vegetable oil in China With half of the supply, China's rapeseed planting area and output rank first in the world, but the yield is lower than in Canada, Australia and other countries. The agronomic traits affecting rape yield mainly include plant height, effective branch number, branch position and flowering time. The number of branches is one of the important plant-type traits related to crop yield. In rapeseed, increasing the number of branches per plant can significantly increase the number of pods in the whole plant, thereby increasing the yield of the individual plants. There was a very significant positive correlation. Plant height is also an important plant type trait of a crop. Properly reducing the plant height can improve the lodging resistance of the crop, increase yield and facilitate mechanized harvesting. The dwarf gene has achieved great success in the "green revolution" in rice and wheat. Rape dwarf traits are extremely important for lodging resistance and improving yield.

Traditional hybrid breeding has played a key role in increasing the yield of oil menu sites in China, but there are also many constraints, such as low seed production efficiency, long breeding time, high labor intensity, and large environmental impacts. Today ’s plant genomes Engineering technology provides more and better options for solving these problems.

CRISPR-Cas9 was originally a defense system for bacteria and archaea to respond to the invasion of foreign viruses. Now it has been widely used in the genetic modification of human cells, mice, zebrafish, rice, etc. It is mainly composed of two elements: the guide sequence sgRNA and Cas9 nuclease. SgRNA can recognize specific sequences in the genome through base complementation, and guide Cas9 nuclease to cut its DNA double strand, resulting in double-strand break (DSB). Homologous recombination (HR) or non-homologous recombination (NHEJ) for DNA repair. This imprecise repair method can generate site-directed mutations of genes, cause gene silencing, and complete targeting. Because the splicing of DNA depends on the pairing of sgRNA with specific sequences in the genome, designing specific targeted sgRNA is the key to avoiding off-target effects.

Although CRISPR technology is a useful tool for gene function research, it can also be used for gene editing to improve crops. However, in polyploid crops such as wheat and rape, the genes are copied in each sub-genome, which greatly improves CRISPR-Cas9. The difficulty of target shooting is only successful if these homologous copies are silenced together. Therefore, higher requirements are imposed on the design of sgRNA: for example, it is necessary to design or concatenate sgRNA target sites in conserved regions of all homologous genes.

Summary of the Invention

The purpose of the present invention is to provide two sgRNAs that specifically target the BnMAX1 gene of Brassica napus. The two sgRNAs are designed in the first exon of the gene, and each sgRNA can mediate A and CRISPR through the CRISPR-cas9 system. The BnMAX1 gene is cut in the C genome to achieve the purpose of gene knockout.

Another object of the present invention is to provide a method for knocking out the BnMAX1 gene of Brassica napus using the CRISPR-Cas9 system and its application. By designing an oligo DNA sequence and a commercial CRISPR-Cas9 vector, a BnMAX1 gene knockout vector can be quickly constructed. Through genetic transformation mediated by Agrobacterium, the mutant strain of BnMAX1 gene knockout obtained by screening can be used for improving rape traits.

In order to achieve the above objective, the present invention adopts the following technical solutions:

A method for specifically knocking out the BnMAX1 gene of Brassica napus using the CRISPR-Cas9 system. The method includes the following steps:

(1) Based on the conserved regions of two copies of the BnMAX1 gene, the DNA sequences of two sgRNAs were designed. Both sgRNAs were located on the first exon of two copies of the BnMAX1 gene, such as SEQ ID NO.1 and SEQ ID NO. 2 shown;

(2) According to the sgRNA sequence and commercial CRISPR-Cas9 vector requirements, design and synthesize two pairs of single-stranded oligo DNA sequences, the sequences of which are shown in SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, SEQ ID ID NO .6;

(3) Two pairs of single-stranded oligo DNA are annealed to form double-strands, and are made into Oligo dimers;

(4) ligating the prepared Oligo dimer with a commercial CRISPR-Cas9 vector, and then transforming E. coli DH5α to obtain two plant expression vectors, named BnMAX1-Cas9-1 and BnMAX1-Cas9-2, respectively;

(5) Agrobacterium-mediated transformation of the BnMAX1-Cas9-1 or BnMAX1-Cas9-2 vector into Brassica napus (such as Kale spring rape 862 and Kale winter rape Zhongzhong 6), in the transgenic offspring Screening of plants with BnMAX1 gene mutations in both A and C chromosomes;

Used to detect the mutations of the two copies of the BnMAX1 gene on the A and C chromosomes: the upstream and downstream nucleotide sequences of the primers for the identification of the A chromosome copy are shown in SEQ ID NO.7 and SEQ ID NO.8, C The upstream and downstream nucleotide sequences of chromosome copy identification primers are shown in SEQ ID NO. 9 and SEQ ID NO. 10;

(6) Determine whether the two copies of the BnMAX1 gene were successfully knocked out based on the amplification results of the identified primers, and select plants with functional mutations in the BnMAX1 protein.

The above method was used to specifically knock out the BnMAX1 gene in Brassica napus to obtain BnMAX1 protein loss-of-function mutation plants. Based on this, further screening of single plants without screening markers was carried out. Phenotypic identification and character inspection showed that homozygous mutant lines The number of branches increased significantly, and the plant height also decreased significantly. The number of fruit per plant and yield increased significantly, which could be used to improve rape traits.

The present invention utilizes the CRISPR-Cas9 system to knock out two copies of the BnMAX1 gene of Brassica napus, and selects two target fragments in the conserved region of the first exon of the two copies of the BnMAX1 gene of Brassica napus and constructs BnMAX1-Cas9 plants respectively. The expression vector was introduced into the hypocotyl of Brassica napus by Agrobacterium infection and regenerated shoots. Under the combined action of two kinds of sgRNA and Cas9 nuclease, both copies of BnMAX1 gene were doubled. Chain shearing, through the cell's own repair function, finally achieves random insertion or random deletion on the target gene fragment, and completes gene knockout.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

The present invention can quickly obtain a CRISPR-Cas9 knockout vector of a target gene through an oligo DNA sequence designed based on sgRNA and a commercial CRISPR-Cas9 vector, simplifying the construction steps and speeding up the research process.

Phenotypic identification and character inspection of the homozygous mutant Brassica napus strains obtained by the present invention show that the number of branches of the strain is significantly increased, the plant height is also significantly reduced, and yield traits such as the number of carobs and yield per plant are significantly increased. The invention provides an efficient breeding method, germplasm resources and theoretical technical support for cultivating high-yield and lodging-resistant rapeseed varieties.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: BnMAX1-Cas9-1 target site

Figure 2: BnMAX1-Cas9-2 target site

Figure 3: Schematic of BnMAX1-Cas9-1 and BnMAX1-Cas9-2 vectors

Figure 4: Nucleotide sequence of the A03 site of the T0 mutant of the BnMAX1-Cas9-1 transformed plant

Figure 5: BnMAX1-Cas9-1 sequencing of the A03 locus of a single mutation in the T0 generation

Figure 6: BnMAX1-Cas9-1 nucleotide sequence of the C03 locus of the single mutant of the T0 generation

Figure 7: BnMAX1-Cas9-1 Sequencing peak of the C03 locus of a single mutation in the T0 generation

Figure 8: BnMAX1-Cas9-2 nucleotide sequence of the A03 locus of the mutant T0 plant

Figure 9: BnMAX1-Cas9-2 sequencing of the A03 locus of a single mutation in the T0 generation

Figure 10: BnMAX1-Cas9-2 nucleotide sequence of the C03 locus of a mutant plant in the T0 generation

Figure 11: BnMAX1-Cas9-2 sequencing of the C03 locus at the T0 generation with a single mutant

Figure 12: Mutation pattern of hygromycin-resistant BnMAX1-Cas9 transformed lines

Figure 13: Phenotype of BnMAX1-Cas9-1-8 transformed lines

detailed description

The following describes the present invention in detail with reference to examples, but is not intended to limit the scope of the present invention. Without departing from the concept of the present invention, modifications or replacements made to the methods, steps or conditions of the present invention belong to the protection scope of the present invention.

The instruments, reagents, materials, etc. involved in the following experimental examples are conventional instruments, reagents, materials, etc. in the prior art unless otherwise specified, and can be obtained through formal commercial channels. Unless otherwise specified, the test methods and detection methods in the following experimental examples are conventional test methods and detection methods in the prior art.

Experimental example 1: sgRNA design of BnMAX1 gene CRISPR-Cas9 in Brassica napus and construction of vectors BnMAX1-Cas9-1 and BnMAX1-Cas9-2

1.sgRNA sequence determination

Brassica napus is a tetraploid crop with two sets of genomes, A and C. The BnMAX1 gene has one copy in each of the two sets of genomes. The two copies were sequenced, and a protoadjacent motif (NGG) motif (NGG) was searched in the conserved region. A 20 bp sequence at the 5 'end of the PAM position was the sgRNA sequence. In the present invention, two sgRNAs are designed, the sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2, and both are located in the first exon, and the target sites are shown in Figs.

2.Synthesis of Oligo DNA single strand

The CRISPR-Cas9 vector construction kit used in the present invention was purchased from Hangzhou Baige Biotechnology Co., Ltd. (Cat # BGK01). Therefore, when designing Oligo DNA single strand, it was synthesized according to the kit instructions:

UPoligo: 5’-TGATTG + sgRNA sequence-3 ’

LOWoligo: 5’-AAAC + sgRNA reverse complement sequence + GA-3 ’

The two pairs of Oligo DNA single-stranded sequences of the present invention are shown in SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, SEQ ID No. 6, and were synthesized by Wuhan Qingke Biotechnology Co., Ltd.

3. Preparation of Oligo Dimer

The synthesized two pairs of Oligo DNA were dissolved in water to 10 μM, mixed according to the following reaction systems, heated at 95 ° C. for 3 minutes using a PCR instrument, and then slowly reduced to 20 ° C. at a rate of about 0.2 ° C./sec.

4.Construct Oligo Dimer to CRISPR-Cas9 Vector

The two groups of Oilo dimer were mixed with each component on ice according to the following reaction system. After mixing, the mixture was reacted at room temperature (20 ° C) for 1 hour.

5. Transformation of E. coli

(1) Add 5 μl of the reaction solution to each of the two groups of reaction systems to 100 μl of DH5α competent cells and mix well;

(2) Allow the ice bath to stand for 30 minutes, avoid shaking during the period, and keep it strictly;

(3) Take out gently, heat shock at 42 ° C for 60 seconds, and immediately place on ice for 2 minutes;

(4) Add 600 μl of LB liquid medium, and resuscitate at 37 ° C and 200 rpm for 1 hour;

(5) Take an appropriate amount of the bacterial solution and coat it on an LB plate containing kanamycin, and invert and culture at 37 ° C overnight.

6. Identification of positive clones and plasmid extraction

Pick out two sets of monoclonal antibodies that grow out of the reaction, inoculate in LB liquid + Kan medium, incubate at 37 ° C and 200 rpm overnight, take half of them to Wuhan Qingke Biotechnology Co., Ltd., and use the sequencing primer provided by the vector kit cas9-F: 5'-CCCAGTCAC GACGTTGTAAA-3 'was identified by sequencing. The correct bacterial solution was identified by sequencing to extract plasmids, and the target vectors BnMAX1-Cas9-1 and BnMAX1-Cas9-2 were obtained. The schematic diagram of the vector is shown in FIG. 3.

Experimental example 2: BnMAX1-Cas9-1 and BnMAX1-Cas9-2 vectors transformed Agrobacterium GV3101

Add 5 μl of BnMAX1-Cas9-1 and BnMAX1-Cas9-2 DNA to 100 μl of GV3101 Agrobacterium competent cells, mix well in an ice bath for 5 minutes, freeze with liquid nitrogen for 1 minute, and incubate at 37 ° C for 5 minutes, add 700 μl Liquid LB medium was cultured at 28 ° C. and shaken at 200 rpm for 4 hours. After the end of the culture, take an appropriate amount of the bacterial solution and spread it on an LB solid dish containing 50 mg / L kanamycin, 50 mg / L gentamicin, and 50 mg / L rifampicin; incubate at 28 ° C for 2 days, and select the monoclonal , Inoculated in LB liquid medium containing kanamycin, gentamicin and rifampicin, cultured at 200 rpm at 28 ° C overnight, and then carried out PCR identification with cas9-F primer and corresponding Lowoligo.

The correct BnMAX1-Cas9-1 and BnMAX1-Cas9-2 Agrobacterium broth were mixed with 50% glycerol and stored at -80 ° C.

Experimental Example 3: BnMAX1-Cas9-1 and BnMAX1-Cas9-2 Agrobacterium Transformed Brassica napus Hypocotyls

1. Explant preparation

Seed surface disinfection using spring rape seed 862 (spring rape seed line collected in the laboratory) and winter rape seed cultivar Zhongshuang 6

(1) Add the seeds to a clean 50 ml centrifuge tube and soak the seeds with 75% alcohol for 5 minutes;

(2) Pour off the alcohol, add 10ml 1.5% liter of mercury, soak the seeds for 15 minutes, and shake them at intervals of several minutes to make the liquid and the seeds fully contact;

(3) Discard the litre of mercury and wash the seeds about 4 times with sterilized single steam;

(4) Dry the remaining water with a pipette, and place the seeds into the M0 medium with tweezers that have been burned and sterilized beforehand, 50 seeds per bottle, and 6 bottles of seeds for each transformation;

(5) 24 degrees, dark culture for 5-6 days.

2. Agrobacterium infection and co-culture

(1) Take 10 μl each of BnMAX1-Cas9-1 and BnMAX1-Cas9-2 Agrobacterium solution stored at -80 ° C, and add it to 1 ml of LB liquid medium containing kanamycin, gentamicin and rifampicin Medium and overnight cultures for activation;

(2) Take out the darkly cultivated seedlings, place them in a large sterilized dish, cut out the hypocotyl with a sterilized scalpel, each section is about 0.8 cm in length, and pour a small amount of DM into the large dish when cutting. Base moisturizing

(3) The activated bacterial solution was connected to 20 ml of LB liquid medium containing kanamycin, gentamicin and rifampicin, and cultured at 28 ° C and 200 rpm until the OD600 value reached 0.4-0.8. Collect the bacterial cells with a centrifuge at 2000 rpm, then add 20 ml of DM medium containing 100 μM AS, resuspend, and shake at 28 ° C for 30 minutes; (4) add the Agrobacterium bacterium solution after DM medium has been cultured for 30 minutes to the cut In a good hypocotyl, infect for 30 minutes, shaking every five minutes or so;

(5) Transfer the explants to an empty petri dish containing sterilized filter paper, spread out, and blot the bacteria solution with small filter paper;

(6) The explants were transferred to M1 medium and cultured in the dark at 24 ° C for two days.

3. Screening and differentiation

The two-day co-cultured explants were transferred to M2 medium containing 5mg / L hygromycin and cultured for about 3 weeks under light. At this time, you can see that the explants became darker and began to swell into callus. The explants were then transferred to M3 medium containing 8 mg / L hygromycin and cultured in a light incubator at a light length of 16 h / d. Subculture was carried out every two weeks to ensure adequate nutrition until seedlings could be differentiated.

4. Rooting

Transfer the calli growing out into the sterilized empty dish, cut the seedlings from the callus with tweezers and a scalpel, and transfer them to the M4 medium light incubator with a light length of 16h / d.

5. Domestication and transplanting

Open and seal the well-rooted bottle seedlings, take out the light incubator, place in the greenhouse for 3-4 days, and then transplant it into the substrate, and put on a clean plastic film to maintain humidity. After 3 days, the holes were gradually opened to prevent wind, so that the plants could adapt to the external environment.

The medium formulation used in this experiment is shown in Table 1.

Table 1 Medium formula

Experimental Example 4: Detection of mutations in transgenic plants

1. Screening and detection of transformed plants

The CT0 method was used to extract the DNA of the transgenic T0 generation plants. Among them, 11 T0 generation plants were obtained from BnMAX1-Cas9-1, and 6 T0 generation plants were obtained from BnMAX1-Cas9-2. PCR amplification was used to detect the transformed plants. Primers used It is: Hyg-120-F: 5'-TGTAGGAGGGCGTGGATATG-3 ', Hyg-971-R: 5'-ACTTCTACACAGCCATCGGT-3', and it is tested whether the TO 0-transformed plants contain a hygromycin selection marker.

2. Mutation detection of BnMAX1-Cas9-1 and BnMAX1-Cas9-2 transformed lines

Since the two copies of the BnMAX1 gene are on the third chromosome of the A subgenome and the third chromosome of the C subgenome, two pairs of primers A03-JC-F: 5'-AACACCCTTCATTACAAACACTCA-3 'and A03-JC-R: 5 were designed. '-ACTACTATCAATGGTTGCCTCCC-3' and C03-JC-F: 5'-GCTTCTCCAGATATTCAGATTT-3 ', C03-JC-R: 5'-CAGTTTCTGTCGTCTTTTCTTA-3' (such as SEQ ID ID NO.7, 8 and SEQ ID ID NO.9, (Shown in Figure 10). After the PCR product is recovered, the T vector is ligated, and the sequence of two copies of the BnMAX1 gene of the BnMAX1-Cas9-1 and BnMAX1-Cas9-2 transformed plants is tested. Sequencing results showed that 5 of the 11 BnMAX1-Cas9-1 transformed plants had insertions or mutations at the A03 site, and 4 had insertions or mutations at the C03 site. The nucleotide sequences of the A03 and C03 sites are shown in Figure 4. As shown in Figure 6, the sequencing peaks are shown in Figures 5 and 7. Of the 6 BnMAX1-Cas9-2 transformed plants, 4 at the A03 site contained insertion or deletion mutations, and 3 had insertions or deletions at the C03 site. Indeed, the nucleotide sequences are shown in Figures 8 and 10, and the sequencing peaks are shown in Figures 9 and 11.

Experimental Example 5: Phenotype observation, trait statistics and non-hygromycin marker screening of transgenic T2 population

After the T0 generation of BnMAX1-Cas9-1 and BnMAX1-Cas9-2 were collected, the T1 generation was continued to be planted. Phenotypic plants were isolated from the two T1 generation lines of BnMAX1-Cas9-1, BnMAX1-Cas9-1-8 and BnMAX1-Cas9-1-11. Among the T1 generation lines of BnMAX1-Cas9-2, three lines of BnMAX1-Cas9-2-2, BnMAX1-Cas9-2-3, and BnMAX1-Cas9-2-5 were found to have phenotypic plants: visual table The type is that the number of branches in the bottom part of the transformed line increases. PCR tests using A03-JC-F / R and C03-JC-F / R primers revealed that all phenotypic mutations were homozygous, that is, two copies of the BnMAX1 gene on the A and C genomes were knocked out except.

At the same time, hygromycin was used to identify primers Hyg-120-F: 5'-TGTAGGAGGGCGTGGATATG-3'Hyg-971-R: 5'-ACTTCTACACAGCCATCGGT-3 'to Tn generation of BnMAX1-Cas9-1-8 and BnMAX1 -Isolation and identification of hygromycin in T1 generation of Cas9-2-2, used to screen for the two copies of the BnMAX1 gene on the A and C genomes that were knocked out and did not have hygromycin resistance. This single plant can be directly used for production because it has no screening resistance marker. One homozygous mutation and no hygromycin resistance were selected from the T1 generation of BnMAX1-Cas9-1-8, and three homozygous mutations and no hygromycin resistance were selected from the T1 generation of BnMAX1-Cas9-2-2. Sexual single plant. The mutation patterns of the BnMAX1-Cas9-1-8 line and the BnMAX1-Cas9-2-2 line are shown in FIG. 12.

After the BnMAX1-Cas9-1-8 homozygous mutant single plant was collected, the T2 generation was continued for character investigation and statistics: 20 BnMAX1-Cas9-1-8 homozygous mutant single plants were planted, and the transformed parent 862 was planted at the same time. In contrast, the phenotype is shown in Figure 13. After the inflorescence at the top of rapeseed is completely opened, the statistical characteristics are as follows: the number of branches and the height of the plant. At the time of harvest, the statistical characteristics are as follows: the number of horns per plant, the length of the horns and the number of horns. After the seeds are dried, the statistics are as follows: Plant yield and thousand kernel weight. Calculate the average value, and the statistical results are shown in Table 2.

Table 2 Table statistical results

According to the above data, it can be seen that the mutant transformant BnMAX1-Cas9-1-8 has a significantly reduced plant height and a significantly increased number of branches compared to its parent. At the same time, the number of carobs and yield per plant have also increased significantly. . The results of seed copying prove that the present invention obtains a functional mutant strain by using the CRISPR / Cas9 system to knock out the BnMAX1 gene of Brassica napus, which can reduce the height of rapeseed and increase the yield at the same time, and provides excellent germplasm for high yield and lodging resistance of rapeseed. Resources and efficient breeding methods and theoretical technical support.

Claims

Two sgRNAs specifically targeting the Brassica napus BnMAX1 gene are characterized in that the nucleotide sequence of sgRNA-1 is shown in SEQ ID NO.1, and the nucleotide sequence of sgRNA-2 is shown in SEQ ID ID NO.2 Show.
A method for specifically knocking out the BnMAX1 gene of Brassica napus using the CRISPR-Cas9 system includes the following steps:

(1) using the sgRNA according to claim 1 as a target sequence, designing and synthesizing forward and reverse oligonucleotide sequences, respectively, annealing to form double strands, and respectively making Oligo dimers;

(2) ligating the Oligo dimer with the Cas9 vector to obtain two plant expression vectors;

(3) transforming any kind of plant expression vector into Brassica napus by means of Agrobacterium tumefaciens;

(4) Screening plants with BnMAX1 gene mutations in both A and C chromosomes in transgenic offspring.
The method for specifically knocking out the Brassica napus BnMAX1 gene by using the CRISPR-Cas9 system according to claim 2, characterized in that the oligonucleotide sequence designed according to sgRNA-1 is shown in SEQ ID NO.3-4, The oligonucleotide sequence designed according to sgRNA-2 is shown in SEQ ID No. 5-6.
The method according to claim 2 for specifically knocking out the BnMAX1 gene of Brassica napus by using the CRISPR-Cas9 system, characterized in that, in step (4), a primer sequence for detecting a mutation of the BnMAX1 gene in the A chromosome is SEQ ID NO. As shown in 7-8, the sequence of the primer for detecting the mutation of the BnMAX1 gene in the C chromosome is shown in SEQ ID No. 9-10.
The type of mutation of a nucleic acid sequence obtained by specifically editing the A chromosome A and C chromosome BnMAX1 genes of Brassica napus by using the CRISPR-Cas9 system according to claim 2, the characteristics of which are shown in Figures 4, 6, 8 and 10.
The application of the sgRNA according to claim 1 or the method according to claim 2 to improving rape traits includes increasing the number of rape branches, increasing the number of carob fruits per plant, reducing rape height, and increasing yield.