Open access peer-reviewed chapter - ONLINE FIRST

Callus Induction from Unpollinated Ovary Explants of Beans

By Asli Küçükrecep, Dilek Tekdal, İlknur Akça, Selim Çetiner and Rüştü Hatipoğlu

Submitted: March 2nd 2021Reviewed: September 10th 2021Published: October 16th 2021

DOI: 10.5772/intechopen.100392

Downloaded: 33

Abstract

Beans one of the essential plant protein sources for human and animal diets. Conventional breeding methods have been used to develop the cultivars of beans with high quality and high yield. However, conventional methods of plant breeding are time-consuming and laborious. Biotechnological methods can accelerate the breeding process in conventional plant breeding. However, the beans are thought to be a recalcitrant crop plant for applying biotechnological methods since plant regeneration under in vitro conditions in beans is not successful. Developing an appropriate method for in vitro bean regeneration remains a significant problem. The objective of this study was to develop a protocol for the culture of unfertilized ovaries of beans. Culture media and genotype are effective on the success of in vitro cultivation. For this reason, 12 genotypes of beans and some nutrient media such as MS and B5 with various 2,4-D/kinetin combinations were tested to obtain callus from unfertilized ovaries. The highest callus induction was obtained with a medium containing 2,4-D (0.5 mg L−1) and Kinetin (2.5 mg L−1). A literature review on beans indicates that no ovary culture has been carried out on tested varieties in this study to date.

Keywords

  • callus
  • common bean
  • 2
  • 4-D
  • Kinetin
  • ovary culture

1. Introduction

Due to the increasing awareness of healthy nutrition globally, individuals obtain most of their daily calorie needs from plant-based foods. Legumes, which constitute the primary source of vegetable protein (22%), have an important place in human nutrition as an alternative to meat products. In addition to their rich nutritional values, legumes are also known as soil friendly due to their ability to bind the free nitrogen of the air to the soil [1]. Beans are one of the most grown edible legume plants in the world. Common bean (Phaseolus vulgarisL. 2n = 2x = 22) is a diploid species with a wide range of variability of phenotypic characteristics due to its tolerance to a variety of agroecological environments [2, 3, 4]. According to 2016 FAO data, the dry bean was grown on an area of 29.392.817 ha worldwide; fresh bean cultivation was carried out on an area of 1.557.233 ha [5]. Turkey is a significant producer of the economically valuable Fabaceae plant family. According to TÜİK data, the most cultivated crop after chickpea and lentil among legumes in Turkey is beans [6]. Although Turkey could not rank in the top 10 worldwide in dry bean production, it is the third largest green bean producer after China and Indonesia in the world (Figure 1) [7].

Figure 1.

Statistical data of dry and green bean production of countries in the world. * From FAOSTAT database, May 2021 [7].

One of the biggest problems encountered in breeding studies of beans with conventional breeding methods is that the breeding process is long. Different molecular marker systems have been developed to shorten this breeding process. In addition, bean growers often use local bean varieties that are available as a population. These populations used are not genetically and physically pure. This situation causes different problems in bean cultivation: (a) mechanized agriculture is complex because individuals in the population do not show uniform growth and development, and (b) problems occur in both cooking and storage of non-uniform products [8]. It is known that the first breeding studies of legumes in Turkey started in 1965 on fresh beans. The bean plant shows the feature of self-fertilization due to its flower structure, and foreign pollination by insects is also possible. There is a flag (vexillum) leaf on the outside of the flower, a fin (alea) at the bottom, and a boat (carina) in the middle of the flower. The flower has 10 stamens, and these are located in the carina.

It is possible to obtain doubled haploid plants by culturing ovaries under in vitroconditions and subsequent chromosome doubling [9]. Although there are many studies on this subject in some plant species, few studies are on obtaining haploid beans [2]. Haploid and doubled haploid plants are currently used in genetic mapping, QTL analysis, mutation breeding, and genomic studies. In addition, homozygosity is achieved in one generation by using doubled plants. Although selfing is possible in the bean plant, it takes a long time to reach homozygous. Crossbreeding can be difficult due to the flower structure. It is known that classical hybridization studies require a high labor force; selfing is required to obtain a pure line and takes a long time, such as 7–9 years. In the dihaploidization method, haploid plants are made doubled haploid as a result of chromosome folding using various chemicals. Each of these 100% homozygous lines obtained is a candidate of a variety.

In this chapter, a protocol for morphogenetic callus induction from ovary samples in beans is defined. This protocol is strongly repeatable for 11 different P. vulgarisgenotypes and Phaseolussp. (1 genotype).

Advertisement

2. Materials and methods

2.1 Plant materials

A total of 12 bean genotypes, seven genotypes selected from local lines and five commercial varieties, were used within the project’s scope. Details on the total 12 bean genotypes are given in Table 1.

NoGenotype/variety nameOrigin and characteristics
1AkmanIt is a variety registered by the Transitional Zone Agricultural Research Institute in 1998. It has a plant height of 60–70 cm. It is a variety with a harvest maturity period of 115–125 days
2Bitlis-76Local bean line, it was selfed three times and made homozygously
3Bitlis-117Local bean line, it was selfed three times and made homozygously
4GöksunIt is a variety registered by the Transitional Zone Agricultural Research Institute in 2012. It has a plant height of 90–100 cm. It is a variety with a harvest maturity period of 104–124 days
5GöynükIt is a variety registered by the Transitional Zone Agricultural Research Institute in 1998. It has a plant height of 45–55 cm. It is a variety with a harvest maturity period of 110–120 days
6Hakkari-12Local bean line, it was selfed three times and made homozygously
7KaracaşehirIt is a variety registered by the Transitional Zone Agricultural Research Institute in 1990. It has a plant height of 50–55 cm. It is a variety with a harvest maturity period of 110–115 days
8ÖncelerIt is a variety registered by the Transitional Zone Agricultural Research Institute in 1990. It has a plant height of 40–50 cm. It is a variety with a harvest maturity of 105–110 days
9Tunceli-1Local bean line, it was selfed three times and made homozygously
10Van-59Local bean line, it was selfed three times and made homozygously
11Small reddish beanIt was obtained from the growers in the town of Elmalı in the province of Niğde in Turkey
12LeklekLocal variety, it was obtained from the grower in the Gülnar district of Mersin Province in Turkey

Table 1.

Information on bean genotypes used in the study.

General views of seeds belonging to 12 bean genotypes are given in Figure 2.

Figure 2.

General images of seeds belonging to 12 bean genotypes (numbering of genotypes as indicated inTable 1).

2.2 Seed viability detection

The 2,3,5 triphenyl tetrazolium chloride method recommended by ISTA [10] was used for seed viability determination. The seeds of the genotypes tested were soaked in water for 24 h and peeled. Then, seeds were taken into 1 g L−1 2,3,5 triphenyl tetrazolium chloride solution, and viability controls were carried out after 24 h.

2.3 Planting seeds and growing plants

Seeds of bean genotypes were sown in the greenhouse. Considering the weed reproduction situation, the seeds were first sown in viols containing peat and perlite (1:1) and allowed to germinate in order for the seeds to germinate easily. Plantlets were developed in viols before they were transferred to pots. Humic fulvic acid was applied with life water in order to remove the initial stress and increase root activity while the plants were transferred to the pots. Plants growing in viols were transferred in large pots. Plants were planted in pots with 20 cm between the rows and plants and 10 plants in each pot.

2.4 Ovary culture

2.4.1 Surface sterilization

Flower buds of an appropriate size determined for the ovary culture experiment should be kept under tap water for 30 min to remove soil and/or dust residues. After then, it was washed several times with sterile distilled water. Explants taken in a sterile cabinet (Demair, class II A2 MSC 120) are rinsed in 70% EtOH for 1 min. Then, they were washed with pure water. Flower buds were kept in 25% NaOCl (sodium hypochlorite) for 15 min. The surface sterilization process of the samples was completed by washing 4–5 times with sterile distilled water. The unpollinated ovaries in the sterilized flower bud were isolated under a stereomicroscope (Olympus SZ61, Japan) and used as the explant in the tissue culture study.

2.4.2 Culture conditions

The sepals and petals of the flower buds, whose surface sterilization has been completed, were carefully removed. The isolated ovaries were then placed on the different basic media (MS and B5) [11, 12]. Different concentrations of 2,4-D (0, 0.5, 1.0, 2.0 mg L−1) and Kinetin (Kin) (0, 0.5, 1.0, 2.0, 2.5, 3.0, 3.5, and 4.0 mg L−1) and their combinations were added to the basic media (Table 2). The experiment was set up with three Petri dishes for each genotype and five ovaries in each petri dish (15 ovaries in total). The samples were kept in styrofoam until callus formation was observed and left to the culture in a climate cabinet (Miprolab, Ankara, Turkey) at 26 ± 1°C.

Experimental plan for ovary culture
Application codeKin (mg L−1)2,4-D (mg L−1)Trial plan for each variety/genotype
MS
1003 Petri dishes (5 ovary explants in each pet)
20.50.53 Petri dishes (5 ovary explants in each pet)
30.513 Petri dishes (5 ovary explants in each pet)
40.523 Petri dishes (5 ovary explants in each pet)
510.53 Petri dishes (5 ovary explants in each pet)
6113 Petri dishes (5 ovary explants in each pet)
7123 Petri dishes (5 ovary explants in each pet)
820.53 Petri dishes (5 ovary explants in each pet)
9213 Petri dishes (5 ovary explants in each pet)
10223 Petri dishes (5 ovary explants in each pet)
112.50.53 Petri dishes (5 ovary explants in each pet)
122.513 Petri dishes (5 ovary explants in each pet)
132.523 Petri dishes (5 ovary explants in each pet)
1430.53 Petri dishes (5 ovary explants in each pet)
15313 Petri dishes (5 ovary explants in each pet)
16323 Petri dishes (5 ovary explants in each pet)
17333 Petri dishes (5 ovary explants in each pet)
183.50.53 Petri dishes (5 ovary explants in each pet)
193.513 Petri dishes (5 ovary explants in each pet)
203.523 Petri dishes (5 ovary explants in each pet)
2140.53 Petri dishes (5 ovary explants in each pet)
22413 Petri dishes (5 ovary explants in each pet)
23423 Petri dishes (5 ovary explants in each pet)
24443 Petri dishes (5 ovary explants in each pet)
B5
25003 Petri dishes (5 ovary explants in each pet)
260.50.53 Petri dishes (5 ovary explants in each pet)
270.513 Petri dishes (5 ovary explants in each pet)
280.523 Petri dishes (5 ovary explants in each pet)
2910.53 Petri dishes (5 ovary explants in each pet)
30113 Petri dishes (5 ovary explants in each pet)
31123 Petri dishes (5 ovary explants in each pet)
3220.53 Petri dishes (5 ovary explants in each pet)
33213 Petri dishes (5 ovary explants in each pet)
34223 Petri dishes (5 ovary explants in each pet)
352.50.53 Petri dishes (5 ovary explants in each pet)
362.513 Petri dishes (5 ovary explants in each pet)
372.523 Petri dishes (5 ovary explants in each pet)
3830.53 Petri dishes (5 ovary explants in each pet)
39313 Petri dishes (5 ovary explants in each pet)
40323 Petri dishes (5 ovary explants in each pet)
41333 Petri dishes (5 ovary explants in each pet)
423.50.53 Petri dishes (5 ovary explants in each pet)
433.513 Petri dishes (5 ovary explants in each pet)
443.523 Petri dishes (5 ovary explants in each pet)
4540.53 Petri dishes (5 ovary explants in each pet)
46413 Petri dishes (5 ovary explants in each pet)
47423 Petri dishes (5 ovary explants in each pet)
48443 Petri dishes (5 ovary explants in each pet)

Table 2.

Medium variants used in ovary culture.

Calli were transferred to fresh MS media without PGR and MS supplemented with thidiazuron (TDZ; 0.4 mg L−1) and salicylic acid (SA; 20 mg L−1) for plant regeneration, which was previously described as a differentiation medium for Phaseolusembryos [13].

2.5 Statistical analysis

Variance analysis was applied to the data on the rate of callus/embryo formation (reaction rate) of ovary explants according to the completely randomized design in split plots with three replications by using MSTAT-C Statistical Program.

Advertisement

3. Results and discussion

The first goal of developing a procedure for indirect regeneration of bean genotypes was to develop an optimum medium for morphogenetic calli induction. In this study, flower buds were used as an explant source. Seed germination occurred in all tested genotypes successfully. Unfertilized ovaries of the genotypes were picked on the day of anthesis. Isolated ovary samples were cultured on 48 different media. For callus induction from the explants, MS and B5 media, including different combinations of auxin (2,4-D) and cytokinin (Kin), were tested. Different concentrations and combinations of Kin (0, 0.5, 1.0, 2.0, 2.5, 3.0, 3.5, and 4.0 mg L−1) and 2,4-D (0, 0.5, 1.0, and 2.0 mg L−1) were investigated to optimize callus induction of 12 bean genotypes. Callus was obtained from all ovary samples studied. Non-morphogenic and morphogenic calli were generated in bean ovary cultures inoculated on different agar media. According to the microscope images of the calli developing from the ovary samples, it was observed that the calli mostly developed at the ends of the cultured ovary sample and had a light yellow-brown color scale (Figures 36).

Figure 3.

Stereo-microscope images (Olympus SZ61, Japan) of callus growing from ovaries cultured in MS medium (black numbers indicate the genotype number and the detail is given inTable 1; red numbers indicate the medium in which callus growth was observed; the detail is given inTable 2; magnification: 1.2×; scale bar 200 μm).

Figure 4.

Calli in petri dishes (black numbers indicate their genotype numbers, and the details are given inTable 1) developed from ovaries cultured in MS medium and given microscopic images inFigure 4; red numbers indicate the medium in which callus development was observed; the detail is given inTable 2.

Figure 5.

Stereo-microscope images (Olympus SZ61, Japan) of callus growing from ovaries cultured in B5 medium (black numbers indicate the genotype number and the detail is given inTable 1; red numbers indicate the medium in which callus growth was observed; the detail is given inTable 2; magnification: 1.2×; scale bar 200 μm).

Figure 6.

Calli in petri dishes (black numbers indicate their genotype numbers, and the details are given inTable 1) developed from ovaries cultured in B5 medium and given microscopic images inFigure 6; red numbers indicate the medium in which callus development was observed, and the detail is given inTable 2.

On the other hand, no regeneration of calli was observed in the samples cultured on the medium free from PGR. The formation of embryos and embryogenic calli was an uncommon occurrence. However, callus was obtained from all 12 varieties tested in this study. Morphogenic calli in the presence of Kin and 2,4-D were characterized by cell proliferation. Nutritional medium with relatively high- and low-growth regulator concentration demonstrated only minor variations in the efficiency of morphogenic calli production. Some factors, such as stress factors and nutrient media composition, are thought to strongly influence the reprogramming of bean megaspore into the sporophytic developmental pathway. Kin was used in the presented study since cytokinins act on bud formation and plant cell division [14]. The effect of Kin and 2,4-D concentration on callus proliferation was observed, and calli increased in size and were nodular and compact (Figures 36).

Unpollinated ovaries/ovules or full flowers can be cultured to produce efficient gynogenesis methods that generate many embryos from female gametic cells. When the literature on haploidization studies conducted with the legume family was examined, very few studies were encountered. In a study on the Cajanus cajanplant, callus and immature embryos were obtained, but it was stated that callus cells initially had haploid and then a large variety of chromosome complements. Also, mature embryos and haploid plants were not obtained [15]. Grewal et al. [16] mentioned that members of the Fabaceae family are recalcitrant and, therefore, the difficulty of their development in culture.

In vitroregeneration and genetic transformation were difficult for P. vulgarisand other members of the Phaseolusgenus since they are recalcitrant. While many in vitroregeneration protocols for P. vulgarishave been published, most of them were related to direct organogenesis or shoot production from meristematic cells [3]. Several reports have been on organogenesis in different cultured explants of P. vulgarishypocotyls, cotyledonary nodes, and embryonic axes [3, 17]. However, no study exists on in vitroembryogenesis from the unpollinated ovary of P. vulgaris.Although plant regeneration is often genotype-specific in tissue culture, callus was successfully obtained from the ovaries of all 12 genotypes in this study. Some plant growth hormones may be stored in the ovary during plant development and may cause a different hormone balance in vitroculture with synthetically added hormones. This situation may also differ within each genotype and cause further growth or developmental problems in the culture. Studies indicate that successful shoot formation is observed in different bean explants cultured in nutrient media where TDZ and IAA are used together [18, 19]. In addition, success has been achieved in media containing a combination of TDZ and IAA in different Phaseolusspecies such as P. acutifoliusA. Gray [20] and P. polyanthusGreenman [21]. Morphogenesis (roots) was induced from ovary samples in this study when the low PGR concentrations were used. Translucent embryos were obtained from ovaries when the calli were transferred to the medium with TDZ (o.4 mg L−1) and SA (20 mg L−1) (Figure 7). When the calli were transferred into the medium free from the PGR, no development was observed, and the calli began to darken. A previous study of Cucumis anguriaL. showed that unpollinated ovaries cultured in vitrodid not enlarge [22].

Figure 7.

Development of heart-shaped embryo on MS medium containing TDZ (o.4 mg L−1) and SA (20 mg L−1) in two weeks (black arrow indicates embryogenic formation; scale bar 200 μm).

According to a literature review, it is known that the B5 [12] medium is more effective in the tissue culture of some Fabaceae family members than the MS medium [2, 23]. Ovary samples taken in culture in MS and B5 media specified in Table 2 were analyzed comparatively for each genotype and each tested medium. The statistically significant interaction of the nutrient medium × genotype revealed that the effect of the nutrient medium on the reaction rate differs depending on the genotypes (Figure 8).

Figure 8.

Callus/embryoid formation rates of ovaries of different genotypes in different nutrient media (%).

The medium, including 2.5 mg L−−1 Kin, provided a significantly higher reaction rate than all other media studied. The reaction rate of the explants differed significantly depending on the genotypes. According to the analysis results, the best callus yield was obtained from B5 media containing 2.5 mg L−1 Kin and 0.5, 1, and 2 mg L−1 2,4-D in the ovary culture experiment. MS medium free from plant growth regulators never triggered callus induction in all tested genotypes, whereas B5 without plant growth regulators resulted in callus induction only in two genotypes (g1 and g6, given in Table 1).

In the future, these findings might act as a clue in generating the whole plants in vitroconditions. The future applications of these bean genotypes hold a great promise as a management tool for obtaining the plants against climatic conditions.

Advertisement

4. Conclusions

The most successful medium for callus induction in ovary culture of P. vulgariswas B5, and the influence of different stages of female gametophyte should be investigated for higher callus induction and plant regeneration in common beans. As a result, the technique we describe has the potential to enhance indirect organogenesis in the future and may serve as the foundation for developing a procedure for P. vulgaris. We believe that the research results discussed here contribute to further studies on in vitroregeneration of common beans. Understanding the role of growth regulators for selective bean genotypes has greatly aided bean regeneration under controlled conditions.

Advertisement

Acknowledgments

The study described here was carried out within the Project (No. 119O003) funded by the Scientific and Technological Research Council of Turkey (TÜBİTAK). The authors are grateful to the Republic of Turkey Ministry of Agriculture and Forestry Mersin Directorate of Provincial Agriculture and Forestry for providing Leklek’s seeds. We would like to thank also Assoc. Prof. Dr. Faheem Shahzad Baloch for graciously supplying common bean seeds indicated by numbers 1-11 in Table 1.

Advertisement

Conflict of interest

The authors declare no conflict of interest.

Advertisement

Appendices and nomenclature

MS

Murashige and Skoog medium

B5

Gamborg’s medium

2,4-D

2,4-dichlorophenoxyacetic acid

Kin

Kinetin

P. vulgaris

Phaseolus vulgarisL.

DOWNLOAD FOR FREE

chapter PDF

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite and reference

Link to this chapter Copy to clipboard

Cite this chapter Copy to clipboard

Asli Küçükrecep, Dilek Tekdal, İlknur Akça, Selim Çetiner and Rüştü Hatipoğlu (October 16th 2021). Callus Induction from Unpollinated Ovary Explants of Beans [Online First], IntechOpen, DOI: 10.5772/intechopen.100392. Available from:

chapter statistics

33total chapter downloads

More statistics for editors and authors

Login to your personal dashboard for more detailed statistics on your publications.

Access personal reporting

We are IntechOpen, the world's leading publisher of Open Access books. Built by scientists, for scientists. Our readership spans scientists, professors, researchers, librarians, and students, as well as business professionals. We share our knowledge and peer-reveiwed research papers with libraries, scientific and engineering societies, and also work with corporate R&D departments and government entities.

More About Us