First Gynogenesis of Vanilla planifolia for Haploid Production and Ploidy Verification Protocol

Abstract

Vanilla orchids are members of the Vanilloideae orchid subfamily, and they hold significant economic value as a spice crop in tropical regions. Despite the presence of 180 known species within this subfamily, commercial production focuses on only three species (Vanilla planifolia, V. odorata, and V. pompona) and one hybrid (V. × tahitensis), prized for their aromatic qualities and bioactive compounds. Limited modern breeding initiatives have been undertaken with vanilla orchids, although recent advancements in genomic research are shedding light on this crop's potential. The protracted breeding cycle of vanilla, coupled with increasing demand for germplasm, underscores the importance of research and breeding efforts in vanilla. This paper outlines a protocol for haploid production in V. planifolia using unfertilized ovaries in tissue culture conditions. Additionally, we present a methodology to confirm the haploid nature of putative haploid lines through stomatal size comparison, chromosome counting, and flow cytometry analysis, proving the successful development of haploid vanilla plants. These findings contribute to the advancement of breeding programs and genetic improvement strategies for the vanilla industry.

Keywords: Vanilla planifolia; gynogenesis; haploids; ploidy; tissue culture.

Figure 1

Hardened putative haploid lines adapted to shade house conditions.

Figure 2

Stomatal morphology size comparison showing significant statistical differences in (A) stomatal length and (B) stomatal width for the putative haploid lines evaluated and the parental control line (AC110); the letter labels on top of the bars refer to the groups generated using Tukey’s HSD test with an alpha value of 0.01.

Figure 3

Stomatal morphology comparison of putative haploid lines and the parent plant with plastids in stomatal guard cells: (A) Stomata from PHaploid#1. (B) Stomata from PHaploid#2. (C) Stomata from PHaploid#3. (D) Stomata from the parent plant (AC110).

Figure 4

Chromosome images: (A) Parent plant (AC110) root tip. (B) Parent plant (AC110) apical meristem. (C) Parent plant (AC110) pollen. (D) PHaploid#1 root tip. (E) PHaploid#2 root tip. (F) PHaploid#3 root tip.

Figure 5

Flow cytometry histograms: (A) Parent plant (AC110) (blue arrow) with wheat as an internal standard (yellow arrow). (B) PHaploid#1 (pink arrow) superposed to the parent plant histogram. (C) PHaploid#2 (pink arrow) superposed to the parent plant histogram. (D) PHaploid#3 (pink arrow) superposed to the parent plant histogram. (E) Parent plant (AC110) (blue arrow) with Barley “Butta 12” as an internal standard (red arrow). (F) PHaploid#1 (pink arrow) superposed to the parent plant histogram. (G) PHaploid#2 (pink arrow) superposed to the parent plant histogram. (H) PHaploid#3 (pink arrow) superposed to the parent plant histogram.

Figure 6

Floral stages used as sources of explants for haploid development. The number was assigned based on the size of the flower bud, with number one being the largest size (unpollinated open flower harvested on the day of opening). (A) Flower bud samples from V. planifolia. (B) Flower bud samples from V. pompona.

Figure 7

Overview of the initiation process for haploid development in vanilla: (A) V. planifolia floral raceme. (B) Closed floral buds. (C) Excised ovary with placental section. (D) Placental ovary section with ovules under stereoscope ×20. (E) Planted placental ovary section in growing medium. (F) Planted placental ovary sections in growing medium. (G) Developing ovaries in placental ovary section in growing medium. (H) Swollen unfertilized ovary excised and planted in growing medium.

Figure 8

Ex vitro adaptation of putative haploid lines: (A) Shoot clusters under O139 media supplemented with BA. (B) Plantlets under O139 media supplemented with liquid coconut endosperm. (C) Fully developed plantlets ready for hardening. (D) A 36-cell tray with a plastic dome for ex vitro adaptation. (E) Plantlets without plastic dome. (F) Adapted putative haploid lines in the shade house.

Figure 9

Temperature and humidity regimen during the hardening process of putative haploid lines. The green line indicates the time when the plastic dome was removed one month after planting.