# Evaluation of Crossability between Nicotiana benthamiana and Nicotiana excelsior

^{1}

^{2}

^{*}

## Abstract

**:**

## 1. Introduction

_{1}hybrid seedlings. In particular, N. benthamiana is one of the two species yielding only viable hybrid seedlings after crossing with N. tabacum [23]. If N. benthamiana can be used as a bridge species to introduce desirable genes from Suaveolentes species to N. tabacum, tobacco breeding will be facilitated. However, while chromosome pairing in intrasectional F

_{1}hybrids of the section Suaveolentes has been well reported, as mentioned in the discussion, crossability between Suaveolentes species has been scarcely reported.

## 2. Materials and Methods

#### 2.1. Plant Materials

#### 2.2. Interspecific Crosses

_{1}seeds were sterilized with 5% sodium hypochlorite for 15 min. The sterilized seeds were sown in Petri dishes (90 mm diameter, 17 mm deep) containing 25 mL of 1/2 MS medium [24] supplemented with 1% sucrose and 0.2% Gelrite (pH 5.8) and then cultured at 25 °C (16 h light/8 h dark; approximately 80 µmol m

^{−2}s

^{−1}). Hybrid seedlings germinated in Petri dishes were potted and cultivated in a greenhouse. The hybrid plants were reciprocally backcrossed with both parents. The number of capsules and hybrids obtained were counted for each cross.

#### 2.3. Analysis of Pollen Viability

#### 2.4. Cytological Analysis of Chromosomes in Meiosis

## 3. Results

#### 3.1. Reciprocal Crosses between N. benthamiana and N. excelsior

#### 3.2. Fertility of Hybrids between N. benthamiana and N. excelsior

#### 3.3. Chromosomal Instability in Reciprocal Hybrids between N. benthamiana and N. excelsior

## 4. Discussion

_{1}hybrids between Suaveolentes species showed full fertility, sterility in the F

_{1}hybrids can be attributed to chromosomal causes [39].

## 5. Conclusions

## Author Contributions

## Funding

## Institutional Review Board Statement

## Informed Consent Statement

## Acknowledgments

## Conflicts of Interest

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**Figure 1.**Leaves and flowers of reciprocal hybrids between N. benthamiana PI 555684 and N. excelsior JT. Scale bars = 1 cm.

**Figure 2.**Pollen grains stained with acetocarmine in N. excelsior JT and a hybrid from the cross N. excelsior JT × N. benthamiana JT. Scale bars = 10 µm.

**Figure 3.**Chromosome instability in hybrids between N. benthamiana JT and N. excelsior JT. (

**a**) Chromosome pairing in meiotic metaphase I. u, univalent; t, trivalent. (

**b**) Chromosomes at meiotic anaphase I. cb, chromosome bridge; lc, lagging chromosome. Scale bars = 10 µm.

Cross Combination | No. of Flowers Pollinated | No. of Capsules Obtained | No. of Seeds Sown | No. of Hybrids Obtained |
---|---|---|---|---|

N. benthamiana JT × N. excelsior JT | 40 | 10 (25% ^{1}) | 178 | 2 (1% ^{2}) |

N. benthamiana JT × N. excelsior PI 224063 | 11 | 2 (18%) | 48 | 0 (0%) |

N. benthamiana JT × N. excelsior PI 555685 | 5 | 1 (20%) | 2 | 0 (0%) |

N. benthamiana PI 555478 × N. excelsior JT | 20 | 0 (0%) | – | – |

N. benthamiana PI 555478 × N. excelsior PI 224063 | 13 | 1 (8%) | 22 | 0 (0%) |

N. benthamiana PI 555478 × N. excelsior PI 555685 | 20 | 1 (5%) | 143 | 0 (0%) |

N. benthamiana PI 555684 × N. excelsior JT | 20 | 2 (%) | 27 | 9 (33%) |

N. benthamiana PI 555684 × N. excelsior PI 224063 | 5 | 1 (20%) | 48 | 0 (0%) |

N. benthamiana PI 555684 × N. excelsior PI 555685 | 20 | 0 (0%) | – | – |

N. excelsior JT × N. benthamiana JT | 18 | 1 (6%) | 32 | 28 (88%) |

N. excelsior JT × N. benthamiana PI 555478 | 20 | 0 (0%) | – | – |

N. excelsior JT × N. benthamiana PI 555684 | 9 | 1 (11%) | 17 | 3 (18%) |

N. excelsior PI 224063 × N. benthamiana JT | 17 | 2 (12%) | 23 | 22 (96%) |

N. excelsior PI 224063 × N. benthamiana PI 555478 | 17 | 2 (12%) | 20 | 11 (55%) |

N. excelsior PI 224063 × N. benthamiana PI 555684 | 6 | 2 (33%) | 20 | 17 (85%) |

N. excelsior PI 555685 × N. benthamiana JT | 3 | 1 (33%) | 31 | 29 (94%) |

N. excelsior PI 555685 × N. benthamiana PI 555478 | 22 | 0 (0%) | – | – |

N. excelsior PI 555685 × N. benthamiana PI 555684 | 2 | 1 (50%) | 34 | 32 (94%) |

^{1}Percentage of capsules obtained.

^{2}Percentage of seed germination.

Cross Combination | Total no. of Pollens Observed | Percentage of Pollen Viability |
---|---|---|

N. benthamiana JT × N. excelsior JT | 368 | 0 |

N. benthamiana PI 555684 × N. excelsior JT | 326 | 0 |

N. excelsior JT × N. benthamiana JT | 348 | 0 |

N. excelsior JT × N. benthamiana PI 555684 | 335 | 0 |

N. excelsior PI 224063 × N. benthamiana JT | 331 | 0 |

N. excelsior PI 224063 × N. benthamiana PI 555478 | 331 | 0 |

N. excelsior PI 224063 × N. benthamiana PI 555684 | 360 | 0 |

N. excelsior PI 555685 × N. benthamiana JT | 313 | 0 |

N. excelsior PI 555685 × N. benthamiana PI 555684 | 318 | 0 |

**Table 3.**Backcrosses of hybrids between N. benthamiana and N. excelsior to both parents as male parents.

Cross Combination | No. of Flowers Pollinated | No. of Capsules Obtained |
---|---|---|

(N. benthamiana JT × N. excelsior JT) × N. benthamiana JT | 20 | 0 |

(N. benthamiana JT × N. excelsior JT) × N. excelsior JT | 20 | 0 |

(N. benthamiana PI 555684 × N. excelsior JT) × N. benthamiana PI 555684 | 20 | 0 |

(N. benthamiana PI 555684 × N. excelsior JT) × N. excelsior JT | 20 | 0 |

(N. excelsior JT × N. benthamiana JT) × N. benthamiana JT | 20 | 0 |

(N. excelsior JT × N. benthamiana JT) × N. excelsior JT | 20 | 0 |

(N. excelsior JT × N. benthamiana PI 555684) × N. benthamiana PI 555684 | 20 | 0 |

(N. excelsior JT × N. benthamiana PI 555684) × N. excelsior JT | 20 | 0 |

(N. excelsior PI 224063 × N. benthamiana JT) × N. benthamiana JT | 20 | 0 |

(N. excelsior PI 224063 × N. benthamiana JT) × N. excelsior PI 224063 | 20 | 0 |

(N. excelsior PI 224063 × N. benthamiana PI 555478) × N. benthamiana PI 555478 | 20 | 0 |

(N. excelsior PI 224063 × N. benthamiana PI 555478) × N. excelsior PI 224063 | 20 | 0 |

(N. excelsior PI 224063 × N. benthamiana PI 555684) × N. benthamiana PI 555684 | 23 | 0 |

(N. excelsior PI 224063 × N. benthamiana PI 555684) × N. excelsior PI 224063 | 20 | 0 |

(N. excelsior PI 555685 × N. benthamiana JT) × N. benthamiana JT | 20 | 0 |

(N. excelsior PI 555685 × N. benthamiana JT) × N. excelsior PI 555685 | 20 | 0 |

(N. excelsior PI 555685 × N. benthamiana PI 555684) × N. benthamiana PI 555684 | 20 | 0 |

(N. excelsior PI 555685 × N. benthamiana PI 555684) × N. excelsior PI 555685 | 20 | 0 |

**Table 4.**Backcrosses of hybrids between N. benthamiana and N. excelsior to both parents as female parents.

Cross Combination | No. of Flowers Pollinated | No. of Capsules Obtained |
---|---|---|

N. benthamiana JT × (N. benthamiana JT × N. excelsior JT) | 20 | 0 |

N. excelsior JT × (N. benthamiana JT × N. excelsior JT) | 14 | 0 |

N. benthamiana JT × (N. excelsior JT × N. benthamiana JT) | 20 | 0 |

N. excelsior JT × (N. excelsior JT × N. benthamiana JT) | 20 | 0 |

N. benthamiana JT × (N. excelsior PI 555685 × N. benthamiana JT) | 20 | 0 |

N. excelsior PI 555685 × (N. excelsior PI 555685 × N. benthamiana JT) | 20 | 0 |

N. benthamiana PI 555684 × (N. excelsior PI 555685 × N. benthamiana PI 555684) | 10 | 0 |

N. excelsior PI 555685 × (N. excelsior PI 555685 × N. benthamiana PI 555684) | 20 | 0 |

**Table 5.**Metaphase I chromosome pairing in reciprocal hybrids between N. benthamiana JT and N. excelsior JT.

Cross Combination | Mean Chromosome Configuration Per Cell | |||
---|---|---|---|---|

Univalent | Bivalent | Trivalent | Tetravalent | |

N. benthamiana JT × N. excelsior JT | 3.50 ± 0.32 | 12.80 ± 0.25 | 2.80 ± 0.18 | 0.12 ± 0.07 |

N. excelsior JT × N. benthamiana JT | 2.80 ± 0.21 | 14.04 ± 0.24 | 2.44 ± 0.15 | 0 |

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**MDPI and ACS Style**

Tezuka, T.; Kitamura, N.; Yanase, M.; Morikawa, T. Evaluation of Crossability between *Nicotiana benthamiana* and *Nicotiana excelsior*. *Agronomy* **2021**, *11*, 2583.
https://doi.org/10.3390/agronomy11122583

**AMA Style**

Tezuka T, Kitamura N, Yanase M, Morikawa T. Evaluation of Crossability between *Nicotiana benthamiana* and *Nicotiana excelsior*. *Agronomy*. 2021; 11(12):2583.
https://doi.org/10.3390/agronomy11122583

**Chicago/Turabian Style**

Tezuka, Takahiro, Naoto Kitamura, Masanori Yanase, and Toshinobu Morikawa. 2021. "Evaluation of Crossability between *Nicotiana benthamiana* and *Nicotiana excelsior*" *Agronomy* 11, no. 12: 2583.
https://doi.org/10.3390/agronomy11122583