Culture Media and Conditions for Pollen Tube Growth in Vitro

To date, various culture media for in vitro pollen tube culture have been developed; some of these are summarised in Table 3. The medium of Brew-baker and Kwack (1963) is one of the most popular for pollen tube culture. Brewbaker and Kwack (1963) tested the pollen of several hundred species, particularly that of petunia and Ornithogalum virens (Liliaceae), to determine a basal medium (Table 3). The media of Hodgkin (1983), Jahnen et al. (1989) and Read et al. (1993) are also popular and have been used as bases from which to develop novel media for biological models. Variations in the media used for Arabidopsis pollen still exist, but the media used for lily

Table 3 Various media used for pollen tube culture*1

Ornithogalum (Liliaceae)

Arabidopsis

tobacco

Original

Brewbaker and

Hodgkin

Li et al.

Derksen

Read

Chen

paper

Kwack 1963

1983 *2

1999

et al. 2002

et al. 1993

et al. 2002

NH4NO3

hno3

100

KNO3

100

100

(0.99 mM)

KCl

75

(1.0 mM)

Ca(NO3)2 ■

300

600

236

4H2O

(2.54 mM)

Ca(NO3)2)

(1 mM)

CaCl2

111

528 (700;

111

700

(1 mM)

CaCl2-2H2O)

(1.0 mM)

MgSO4 ■

200

217

96 (0.8 mM

409 (200;

7H2O

(0.88 mM)

MgSO4)

MgSO4)

Mg(NO3 )2 ■

6H2O

MgCl2

KH2PO4

K3PO4■

H2O

MnSO4 ■

169

4H2O

(1 mM)

ZnSO4 ■

7H2O

H3BO3

100

100

100

100

99.2

100

(1.62 mM)

(1.6 mM)

CUSO4■

(30 ^M

5H2O

CUSO4)

Na2MoO4 ■

2H2O

Sucrose

10%

20%

18%

20%

5%

2%

(585 mM)

Polyethylene-

3%

12.5%

15%

glycol (MW)

(4000)

(6000)

(3350)

Casein

300

MES

15 mM

20 mM

TAPS

20 mM

pH

8 (NaOH)

7

5.9 (KOH)

6

Agarose

0.5%

0.7%

(Bacto-Agar)

Rifampicin

10

pollen have been improved over many years and have almost converged (Table 3).

At least three substances, plus water, are required in the pollen tube culture medium. The first substance is calcium, which is necessary for pollen tip growth (see Hepler et al., this volume). Calcium is usually added to the medium at 10-600 mg/L as nitrate or chloride salts. The second substance is borate, which may be necessary for cell wall formation (Loomis and Durst 1992). Borate is usually added as boric acid, and a concentration of 100 mg/L

Table 3 (continued)

Nicotiana lily alata

Agapanthus

corn poppy

Torenia

Original

Jahnen

Holdaway-

Kim et al.

Prado

Malho and

Franklin-

Higashi-

paper

et al.

Clarke et al.

2003

et al.

Trewavas

Tong et al.

yama et al.

1989

2003 *3

2004

1996

1988

1998*4

NH4NO3

80

HNO3

kno3

100

100

125

(0.99 mM)

KCl

7.5

75

200

(0.1 mM)

(1.0 mM)

Ca(NO3)2 ■

700

300

500

4H2O

(1.27 mM)

CaCl2

11.1

55.5

200

272 (360;

(0.1 mM)

(0.5 mM)

CaCl2 -2H2O)

MgSO4 ■

200

125

7H2O

Mg(NO3 )2 ■

100

6H2O

MgCl2

200

KH2PO4

125

K3PO4■

100

H2O

MnSO4 ■

3

4H2O

ZnSO4■

0.5

7H2O

H3BO3

100

99.2

10

99.2

100

100

10

(1.6 mM)

(0.162 mM)

(1.6 mM)

CUSO4■

0.025

5H2O

Na2MoO4 ■

0.025

2H2O

Sucrose

2%

7%

10%

6%

2.5%

12%

1%

Polyethy

15%

13%

lene gly

(4000)

(4000)

col (MW)

Casein

500

MES

15 mM

0.05 mM

TAPS

pH

6 (KOH)

5.2

6.0

6.0

5.8 (without

adjustment)

Agarose

1.0%

0.4%

1.5%*5

Rifampicin

Units are mg/L unless otherwise noted. *2 Originally established for pollen tube culture in Brassica oleracea but often used for pollen tube culture in Arabidopsis.

*3 Established for pollen tube culture in Lilium formosanum.

*4 The 4% sucrose is now replaced by 13% PEG 4000 to improve the fertilization frequency as described in Sect. 13.2.

*5 Ultra-low gelling temperature agarose.

appears sufficient for most flowering plants. The third substance is sucrose, which is necessary to adjust the osmotic pressure and may be used as a carbon source for respiration and the synthesis of starch, lipids, amino acids and nucleic acids (Vasil 1987). Sucrose is the most suitable sugar source in most flowering plants (Vasil 1987). The optimum osmotic pressure differs among biological models; for example, the optimum sucrose concentration is 10-20% for Arabidopsis (Hodgkin 1983; Li et al. 1999; Derksen et al. 2002; Schreiber and Dresselhaus 2003), 9-18% for tobacco (Cheung et al. 2002; Ro-magnoli et al. 2003), 5-10% for lily (Vidali et al. 2001; Holdaway-Clarke et al. 2003; Kim et al. 2003; Prado et al. 2004), 2.5% for Agapanthus (Malho and Tre-wavas 1996), 11% for maize (Walden 1993), 20% for rice (Kariya 1989), 12% for corn poppy (Franklin-Tong et al. 1988) and 5% for Torenia (Higashiyama et al. 1998).

In addition, other inorganic ions (e.g., K+, Mg2+; Brewbaker and Kwack 1963), buffers to control pH (e.g., 2-Morpholinoethanesulfonic acid, monohydrate (MES); Tupy and Rihova 1984) and organic compounds (e.g., amino acids, casein hydrolysate; reviewed by Vasil 1987) are used in the media to promote pollen tube germination and growth, depending on the plant species. The frequency and timing of the generative-nucleus division also depended on the chemical composition of the medium in tobacco (Read et al. 1993). It is noteworthy that polyethyleneglycol (PEG) 4000-8000 has a dramatic effect on pollen tube germination and growth (Zhang and Croes 1982; Jahnen 1989; Read et al. 1993; Barinova et al. 2002), although the physiological action of PEG is unknown. PEG has been widely used in pollen tube culture media for Arabidopsis, tobacco, Torenia and snapdragon (Barinova et al. 2002; Schreiber and Dresselhaus 2003; Table 3). In Torenia, PEG 4000 increased the viability of both the pollen tube and the naked embryo sac, whereby the frequency of pollen tube attraction in vitro increased fourfold (Higashiyama et al. 2000). However, high concentrations of PEG tend to precipitate in solid media. Thus PEG is sometimes replaced by sucrose when used in solid media (e.g., Cheung et al. 2002). Osmotic potential of 15% PEG 4000 (327 mOsm/kg) corresponds to that of 9% sucrose (325 mOsm/kg) (Jahnen et al. 1989). In the medium for Torenia, 13% PEG 4000 in 1.5% ultra-low gelling temperature agarose is the maximum concentration that balances the effect of PEG 4000 and the extent of precipitation (Table 3). As in cell fusion experiments, PEG appears to remain effective for only one month after its dissolution in water, and its effect is also lost by autoclaving.

The effectiveness of simple media should be tested first. Environmental conditions, such as humidity and aeration, and physiological conditions of the flower are critical and can be tested using a simple medium. Pollen germination may also depend on the population effect, i.e., the pollen concentration. Phytosulphokine (PSK), a peptide hormone of flowering plants, is an intercellular signalling molecule (Chen et al. 2000). It should also be noted that Na+ strongly inhibits pollen tube growth in most flowering plants. Thus, KOH, rather than NaOH, is recommended to adjust the pH. The optimal temperature for pollen tube culture is usually 20-30 °C, and the growth rate of pollen tubes depends on the temperature.

The medium for the in vitro Torenia system (Higashiyama et al. 1998, 2001) is described in Table 3. Torenia pollen tubes grow well in a simple medium containing 300 mg/L Ca(NO3)2 ■ 4H2O, 100 mg/L H3BO3, 1% sucrose and 13% PEG 4000 (Fig. 1), but this medium does not support the culture of ovules that have a naked embryo sac. Therefore, Nitsch's medium (1951), originally established for the culture of excised ovaries, was modified for the cultivation of both the pollen tube and ovule together.

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