Phalaenopsis pollinia storage at sub-zero temperature and its pollen viability assessment

Abstract

Background: In a breeding program, usually only superior parents are chosen for cross hybridization. Pollens of elite cultivars may not be available at hand. Properly stored pollens provide an opportunity for cross hybridization at unavailable time.

Results: Pollen of a Phalaenopsis hybrid was evaluated for the storage ability at different temperatures, including room temperature, 4, - 20, and - 80 °C for up to 96 weeks. The viability of pollen was assessed by TTC staining, in vitro germination and hand pollination during and after storage. Pollen stored at all temperatures for 4 weeks remained viable and capable of successful pollination. Pollen lost its viability after 4 weeks at room temperature. Pollen remains viable after 40 weeks at 4 °C, and after 96 weeks at both - 20 and - 80 °C of storage. Viable pollen could be successfully pollinated to the female parent at all effective storage conditions and produced seeds.

Conclusions: Our results indicate that Phalaenopsis pollen can be stored at 4 °C up to 40 weeks for short-term purpose. For long-term storage, pollen can be kept at both - 20 and -80 °C.

Keywords: Hand pollination; In vitro germination; Low temperature storage; Pollen viability.

Fig. 1

Flower developmental stages (A) and their corresponding pollen germination rate (B) in Phalaenopsis Little Gem Stripes. A Flower developmental stages are defined as: (1) tight floral bud; (2) sepal half-open; (3) flower half-open; (4) prior to anthesis; (5) flower fully open; (6) 1 day after fully open. B In vitro pollen germination of P. Little Gem Stripes was evaluated by incubating in Brewbaker and Kwack medium for 7 days at 25 ± 1 °C and then by Alexander staining. Bars represent mean ± SE, n = 4. Pollen germination rank was represented by ranks with area of pollinia stained: (0) no-germination; (1) 1–20%; (2) 21–40%; (3) 41–60%; (4) 61–80%; (5) 81–100%

Fig. 2

The pollen viability of Phalaenopsis Little Gem Stripes stored at different temperatures as evaluated by TTC staining. RT, room temperature. Scale bar = 5 mm

Fig. 3

Pollen germination rate of Phalaenopsis Little Gem Stripes after storage at four temperatures. In vitro pollen germination of P. Little Gem Stripes was evaluated by incubating pollinia in Brewbaker and Kwack medium after 7 days and then stained. Bars represent mean ± SE, n = 4. Pollen germination rank was represented by ranks: (0) no-germination; (1) 1–20%; (2) 21–40%; (3) 41–60%; (4) 61–80%; (5) 81–100%

Fig. 4

Pollen tubes of Phalaenopsis Little Gem Stripes grown in stigma and ovary cavity of P. Sogo Vivien ‘F858’ after storage. a–d The pollen taken from storage for 96 weeks at four temperatures was pollinated for 7 days. a room temperature storage; b 4 °C; c − 20 °C; d − 80 °C. e Pollen from storage at − 20 °C was pollinated for 35 days, and f for 70 days, g partially magnified from e. Arrow indicates pollen tube (c, d, and f). P, pollinia; O, ovules. Scale bars = 50 μm