Hybridization between diploid and tetraploid parents in sour citrus breeding

Document Type : Full Paper

Authors

• Elham Jalilzadeh ¹
• Asad Asadi Abkenar ²
• Parisa Jonoubi ³
• Sahar Bohlouli Zanjani ⁴
• Reza Fifaei ⁵
• Abuzar Hashempour ⁶

¹ Department of Plant Sciences, Faculty of Biological Sciences, Kharazmi University,Tehran, Iran.

² Department of Molecular Biology, Agricultural Biotechnology Research Institute of North Region, Rasht,, Iran

³ Department of Plant Sciences, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran.

⁴ Department of Molecular Biology, Agricultural Biotechnology Research Institute of North Region,,, Rasht, Iran

⁵ Department of Genetics and Breeding, Citrus and Subtropical Fruits Research Center, Horticultural Science Research Institute, Agricultural Research Education and Extension Organization (AREEO), Ramsar, Iran

⁶ Department of Post-harvest Physiology and Technology, Citrus and Subtropical Fruits Research Center, Iranian Horticultural Sciences Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Ramsar, Iran.

Abstract

Keywords

• Breeding
• lemon
• lime
• seedlessness
• triploid

Main Subjects

• Fruit Science

Extended Abstract

Introduction

              Acid limes and lemons belong to a group of citruses known as acid citrus. Fruits of this group are highly juicy, acidic, rich in vitamin C, and usually seeded. One of the most important goals in citrus breeding is to develop cultivars that produce seedless fruits. Like other citrus fruits, seedless lime and lemon cultivars are highly valuable in both the fresh fruit market and the processing industry. In citrus, fruits of diploid and tetraploid genotypes are generally seeded, whereas only triploid genotypes are seedless. To obtain triploids, tetraploid cultivars must first be produced, and then triploid cultivars can be generated through crosses between diploid and tetraploid plants, or vice versa. The aim of this research was to obtain triploid acid citrus genotypes through controlled crossings between diploid and tetraploid citrus cultivars.

Materials and Methods                                                                                                                                               

In the Agricultural Biotechnology Research Institute of Northern Iran (Rasht), polyploid genotypes of ‘Mexican’ or key lime (Citrus aurantifolia Swingle) and sweet lime (Citrus limettioides Tan.) were previously developed by colchicine treatments and were well preserved. In the present study, these genotypes were used to produce triploid plants.  In the first step, the polyploidy level stability of two key lime genotypes (A and B) and three sweet lime genotypes (C, D, and E) was evaluated using flow cytometry. Additionally, several vegetative and reproductive traits of trees A and B were assessed, including leaf characteristics (blade length, width, and diameter, petiole length, and stomatal density) and fruit traits (fruit weight, length, and width, total soluble solids, total acidity, peel thickness, seed number, juice content, and seed polyembryony).  In the second stage of the study, controlled crosses were performed at the citrus research station in Ramsar between clementine mandarin and ‘Mexican’ lime (genotype B). Furthermore, at the Kotra citrus station (Mazandaran Province), controlled crosses were carried out between three female lemon parents (‘Eureka’, ‘Cook Eureka’, and ‘Lisbon’) and two sweet lime tetraploids (C and D).  For rescue of triploid embryos, seeds were extracted from the fruits of the crosses, washed, and sorted based on their size and weight. Only seeds weighing less than 0.09 g (very small seeds, presumed to be highly monoembryonic) were cultured on B5 medium supplemented with 3% sucrose, 0.7% agar, 0.5 g/L malt extract, and 1 mg/L GA₃.

Results and Dicussion

In tree A, four of the five main branches (A1, A2, A3, and A4), each with a primary flow cytometric peak, were diploid (2n=2X). Only one main branch (A5) exhibited two distinct peaks, indicating a mixoploid (2X+4X) status.  In tree B, all seven main branches were uniformly triploid (2n=3X). The leaves of tree B (triploid) were larger and thicker compared to those of tree A (mainly diploid, except A5), with shorter petioles and more scattered stomata. Fruits from one of the branches in tree B (B4) contained an average of two seeds. Since tree B is triploid and its seedlessness holds commercial value, it is essential to propagate each of its branches vegetatively for maintenance, further study, and use in breeding programs.  Crosses between clementine mandarin and ‘Mexican’ lime produced no fruit or seeds. However, crosses involving three lemon female parents (‘Eureka,’ ‘Cook Eureka,’ and ‘Lisbon’) with tetraploid sweet limes resulted in fruit production. The embryos derived from these crosses, cultured in B5 medium supplemented with 1.5 mg/l GA3, showed the highest germination percentage and shortest germination time.  Thirty-five days after in vitro germination, the seedlings were transferred to ex vitro conditions. Of the 15 seedlings analyzed for ploidy via flow cytometry, ten were diploid (nucellar), and five were triploid (2n=3X). Specifically, four seedlings originated from the ‘Eureka’ × sweet lime cross, and one from the ‘Cook Eureka’ × sweet lime cross.  Future efforts should focus on further studying these triploid seedlings, especially their fruit characteristics, to evaluate their potential as new citrus cultivars for commercial production.

Conclusion

    From crosses between diploid lemon cultivars ‘Eureka’ and ‘Cook Eureka’ (female parents) and tetraploid sweet lime (male parent), a total of five triploid genotypes were obtained. These genotypes should be further evaluated for their phenotypic characteristics in future studies.  For this purpose, we suggeste that the triploid genotypes from this study be propagated onto sour orange or citrumelo rootstocks to ensure their preservation and to produce sufficient plant material for further research

Author Contributions

1. Jalilzadeh and A. Asadi Abkenar conceived and planned the experiments. S. Bohlouli Zanjani,   A. Hashempour, E. Jalilzadeh  and  A. Asadi Abkenar  carried out the experiments. E. Jalilzadeh  and  A. Asadi Abkenar analyzed data. Elham Jalilzadeh  and  Asad Asadi Abkenar wrote the first manuscript. Parisa Jonoubi and Reza Fifaei contributed to the interpretation of the results. All authors provided critical feedback and helped shape the research, analysis and manuscript.

Data Availability Statement

Data available on request from the authors.

Acknowledgements

The authors would like to thank the members of postharvest laboratory of Citrus and Subropical Research Institute of Iran (Ramsar), especially K. Najafi Hir (Retiried) and E. Seyedghasemi.

Ethical considerations

The authors avoided data fabrication, falsification, plagiarism, and misconduct.

Conflict of interest

The author declares no conflict of interest.