Journal of Experimental Botany, Vol. 52, No. 357, pp. 851-855,
April 15, 2001
© 2001 Oxford University Press
Development of an improved medium for germination of Cajanus cajan (L.) Millsp. pollen in vitro
Division of Genetics, Indian Agricultural Research Institute, New Delhi 110 012, India
Received 7 April 2000; Accepted 12 October 2000
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Abstract |
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A simple, reliable medium for pollen germination of Cajanus cajan was developed by modifying Brewbaker and Kwack (BK) medium. Past attempts of C. cajan pollen germination in artificial media were not successful. A medium containing polyethylene glycol 4000 (PEG) showed more than 90% germination for C. cajan var. Pusa 33 only when the young buds (36 h before anthesis) were kept in pollen germination medium (PGM) for 36 h before pollen extraction. Supplementation of PGM with
-amino caproic acid (EACA), an amino acid, showed improved pollen germination in Pusa 33 and also helped to avoid preconditioning of young buds before pollen extraction. It was also observed that there is a genotypic difference in the level of EACA required for in vitro pollen germination. Thus a complete medium for C. cajan genotypes consists of 37.5% sucrose+ 15% PEG 4000+250 mg l-1 boric acid+300 mg l-1 calcium nitrate+100 mg l-1 potassium nitrate+ 200 mg l-1 magnesium sulphate+1% agar+EACA (0, 100, 250, 500, 750 or 1000 mg l-1).
Key words:
Cajanus cajan, in vitro pollen germination, pigeonpea, polyethylene glycol (PEG), -amino caproic acid (EACA).
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Introduction |
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In vitro pollen germination is one of the most convenient and reliable methods used to test the viability of fresh or stored pollen. It is a valuable tool to address basic questions in sexual reproduction. The media used for in vitro germination of pollen of different species ranges from simple sucrose/boric acid media (Linskens, 1967
) to complex media containing polyethylene glycol (Zhang and Croes, 1982; Shivanna et al., 1997) and various amino acids (Read et al., 1993). A medium has been developed that has been widely used and found to be suitable for more than 86 plant species (Brewbaker and Kwack, 1964).
Legume pollen is normally difficult to germinate on artificial medium. In vitro pollen germination in pigeonpea has been reported previously (James et al., 1987; Singh et al., 1992) and a maximum of 49% germination was reported. There is a need to develop a suitable medium to improve the frequency of pollen germination and the quality of pollen tubes that grow in vitro. An optimal medium that enables at least 90% pollen germination would be useful for both basic studies on pollen function and applied aspects such as in vitro pollination/fertilization (Zenkteler, 1990), pollen storage (Barnabas and Kovacs, 1997), male gametophytic selection (Sari-Gorla and Frova, 1997), and wide hybridization (Zenkteler, 1990).
-Amino caproic acid (EACA) is an amino acid. It is derived from n-caproic acid (n-hexanoic acid), a saturated 6 carbon fatty acid (C6H12O2, CH3(CH2)4-COOH) which occurs in milk fats. EACA has been used to improve solubilization of membranes. It has been used in studies on overcoming self incompatibility and incongruity in Phaseolus (Nagarajan and Walton, 1988) and Vigna (Baker et al., 1975; Chen et al., 1978). While EACA has been used as an aqueous spray on young buds or injected into internodes to overcome incompatibility, it has not been used as a constituent of pollen germination medium.
The objective of this study was to develop a suitable medium to improve in vitro pollen germination in pigeonpea. To achieve this, -amino caproic acid was incorporated for the first time into the pollen germination medium.
This study emerged as a part of programme on wide hybridization in pigeonpea (Jayaprakash, 1998). The cross C. volubilisxC. cajan did not yield any seed and pollen pistil studies showed that C. cajan pollen did not germinate on C. volubilis stigma. Different methods to overcome pre-fertilization barriers were used, but pollen did not germinate. In order to carry out in vitro pollination and fertilization of C. volubilis ovules, it was necessary to develop a medium in which pollen would germinate and possibly effect fertilization.
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Materials and methods |
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Cajanus cajan var. Pusa 33 was used for in vitro pollen germination. The seeds were sown in June and most of the in vitro pollen germination studies were carried out during September and March of 1997–99 at the Indian Agricultural Research Institute, New Delhi, India. During these months there is peak flush of flowering when many flowers are borne but only 10–15% flowers set pods. In November, very few flowers are borne.
Pollen germination media (PGM)
In this study, Brewbaker and Kwack (BK) medium (Brewbaker and Kwack, 1964) (10% sucrose, 100 mg l-1 boric acid, 300 mg l-1 calcium nitrate, 200 mg l-1 magnesium sulphate, and 100 mg l-1 potassium nitrate) was used. In a preliminary investigation, media having Brewbaker and Kwack medium salts, different concentrations of sucrose (10, 20, 30, 40%) and/or 15% polyethylene glycol 4000 were screened to select a basal medium. The selected basal medium was then used in subsequent studies and further improved by altering the concentrations of sucrose, boric acid and calcium nitrate one by one, respectively. Since liquid medium gave inconsistent results 1% agar was added in all the media used and the results were consistent in replications.
Pollen collection and culture
Pollen was collected and cultured according to the method described earlier (Singh et al., 1992). Five replicates were used for each medium combination/treatment. All observations on pollen germination and pollen tube growth were taken 3 h after incubation. At least 250 pollen was counted from 5–10 random fields. A pollen grain was considered germinated if pollen tube length was greater than the diameter of the pollen grain. Similarly, 50–60 pollen tubes were measured (using an ocular micrometer) to calculate mean pollen tube length. Budding refers to pollen grains where pollen tubes do not grow longer than the diameter of pollen grain. Incubation temperature was maintained at 20.5±2 °C as this temperature was found to be optimum for germination of pigeonpea pollen.
Pretreatment of young buds
Young buds refer to flowerbuds 36 h or 12 h before anthesis. These unopened buds have receptive stigma and undehisced anthers. At this stage, anthers are yellow and pollen is binucleate with a fully developed exine.
Different pretreatments were given to these young buds at 20.5 °C: (a) pre-hydration in a Petri plate lined with moist filter paper (>90% RH) for 36 h; (b) agarified medium containing 37.5% sucrose in (a) for 36 h; (c) agarified medium containing 37.5% sucrose+15% PEG in (a) for 36 h; (d) pollen germination media (PGM) in (a) for 12 h; (e) pollen germination media (PGM) in (a) for 36 h. Pollen was collected from buds which underwent these treatments and pollen germination tests were carried out in the media G1–G5 (Table 1).
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Supplementation of pollen germination medium with -amino caproic acid (EACA)
In the pollen germination medium G2 (37.5% sucrose, 250 mg l-1 boric acid, other BK salts, 15% PEG, 1% agar) different concentrations of EACA (100, 250, 500, 750, 1000 mg l-1) were added and the resultant media were designated as P1–P5, respectively. In addition to Pusa 33, pollen of seven other randomly selected pigeonpea cultivars was tested on P1–P5 media for germination. Medium G2 was maintained as control in each case.
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Results |
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Budding was observed only in the medium with 40% sucrose. At concentrations below 40%, bursting of pollen grains occurred. On changing the concentration of boric acid and calcium nitrate, a maximum of 43.80% pollen germination was observed in a medium containing 40% sucrose, 250 mg l-1 boric acid and other BK salts. The other combinations showed lesser pollen germination and more bursting.
Addition of PEG improved pollen germination. The medium with 30% sucrose, 15% PEG and BK salts showed over 45% germination with severe pollen and pollen tube bursting. Increasing sucrose concentration to 37.5% controlled pollen bursting substantially. This medium was designated as G. Then other constituents of this medium were altered to improve pollen germination. A set of five media (G1–G5) with different permutations and combinations of boric acid and calcium nitrate showed an improved pollen germination of 53–87% (Table 1
, A). However, pollen and pollen tube bursting was a common feature in all these media used.
Pretreatment of young buds
Pollen obtained from individual anthers just before dehiscence also showed 53–87% germination along with pollen and pollen tube bursting. Another source of pollen, obtained from excised young buds kept in PGM until anther dehiscence, showed some interesting results. In treatments a–c (see Materials and methods) almost all pollen grains germinated but the pollen tubes burst immediately. These pollen grains were considered as non-germinated. Pollen obtained from excised young buds kept in PGM for 12 h (treatment d) and 36 h (treatment e) showed improved pollen germination. The pollen extracted from the in vitro grown buds were germinated in media G1–G5. The pollen obtained after 12 h incubation of young buds in PGM (treatment d) showed improved germination over control (Table 1, B). The media G2 and G3 showed more than 90% germination, but bursting and irregular pollen tube growth indicate that this pre-conditioning of young buds did not favour pollen germination. Pollen extracted after 36 h incubation (treatment e) gave over 91% germination in media G2 and G3 (Table 1 C). It is important to note that the maximum number of smooth pollen tubes (mean tube length 15.64 µm) was observed only in G2 (Table 2). The medium G2 was therefore found to be the most suitable for incubation of young buds before the germination test as the other media showed curled pollen tubes and bursting. The maximum pollen germination as high as 91.85% was observed when excised young buds (36 h before anthesis) of Pusa-33 were kept in PGM-G2 for 36 h and then pollen was taken out and germinated. All the above germination studies were carried out on pollen obtained at the peak flowering stage (September and March).
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On the other hand, in November when plants bear very few flowers the pollen showed above 95% germination (95.63±3.502) in medium G2. This pollen did not require any pretreatment of young buds for optimum germination. Thus there was a distinct seasonal effect of the source of pollen on pollen germination. This prompted us to carry out all subsequent studies on the September-borne flowers, i.e. at peak flowering stage to mimic the November effect.
Effect of EACA on pollen germination
To avoid preconditioning of young buds before the pollen germination test, EACA was added in PGM-G2. The media P4/P5 containing 750 or 1000 mg l-1 EACA gave >91% pollen germination as well as smooth pollen tubes when Pusa-33 was used. In order to confirm the effect of EACA on pollen germination, some other genotypes were also included (Table 3). In the control medium devoid of EACA (G2), 0–98% germination were recorded. Addition of EACA to PGM-G2 further increased the percentage pollen germination of genotypes that showed poor germination in control (G2), but the requirement of EACA varied with genotypes (Table 3). The genotype IDTSP51, invariably showed >98% germination with normal smooth pollen tubes in all the media. It is evident from Table 3 that some genotypes showed as low as 68.5% pollen germination even after the addition of EACA. Germination rate in these genotypes can be improved by manipulation of the media constituents especially boric acid and/or calcium nitrate. Table 4 shows the mean pollen tube length of two genotypes Pusa 33 and IDTSP51 at different time intervals on medium P2. Pigeonpea pollen initiate germination only about 2 h after incubation (Fig. 1a). The mean pollen tube length in these genotypes was 27–33 µm at 3 h (Fig. 1b), 45–53 µm at 4 h (Fig. 1c) and 78 µm at 5 h, respectively.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In the present study, Brewbaker and Kwack's medium (Brewbaker and Kwack, 1964
) with some minor modifications was used for in vitro pollen germination of pigeonpea. The pollen germination medium (PGM) was also supplemented with PEG 4000 and/or EACA. Use of a liquid medium consisting of 40% sucrose, 250 mg l-1 boric acid and 200 mg l-1 calcium nitrate to germinate pigeonpea pollen was reported earlier for C. cajan, but only 48.7% pollen germinated (James et al., 1987). Later Singh et al. modified the same medium by adding 0.7% agar and achieved a maximum of 43.1% pollen germination at 22 °C (Singh et al., 1992). In the present study a set of media with PEG were tried. In these media 53–87% germination was observed with profuse pollen and pollen tube bursting. Addition of PEG improved the percentage germination in pigeonpea. It has also been found useful in another legume, chickpea (Shivanna et al., 1997). To control bursting of pollen and pollen tubes, several pretreatments were used. Heat treatment (pollen was kept in an oven at 40 °C for 30 min or 60 °C for 20 min) did not improve percentage germination. A similar effect was also obtained upon prehydration of pollen. Another treatment gave improved response. The pollen obtained from excised young buds (36 h before anthesis), kept in PGM-G2 showed >91% germination. Pigeonpea produces lot of flowers but the inherent pod set is only 10–15% of the bloom (Pandey and Singh, 1981). Pollen germination studies from two different months revealed that the fewer flowers there were on the plant, the greater the germination percentage. This indicates that at peak flowering stage nutrients may be a limiting factor during pollen maturation and when supplied externally (as in the bud pretreatment) pollen germination rate improved.
Further, the medium G2 was improved by the addition of EACA, which helped to avoid the pretreatment of young buds before pollen extraction. It was noticed that there are genotypic differences for pollen germination. Among the genotypes tested, IDTSP51 alone did not require EACA, but for others the requirement varied. Thus pollen germinability of any genotype of pigeonpea can easily be checked using the PGM-G2 with or without EACA. This is the first time EACA has been used as a constituent of pollen germination medium. Earlier EACA was used to obtain interspecific hybrids in some legumes (Baker et al., 1975; Chen et al., 1978; Nagarajan and Walton, 1988). These authors reported that EACA enhanced the hybrid embryo growth significantly. EACA is an amino acid, a derivative from n-caproic acid, a saturated 6-carbon fatty acid, which occurs in milk fats. It has recently been shown that lipids present in the stigma are involved in directional pollen tube growth (Arts et al., 1998). Though the mode of action of EACA in promoting pollen germination is not clear, it is possible that it does so via membrane solubilization thus increasing lipid or nutrient availability.
The optimal medium developed in this study can be used effectively for experiments on in vitro pollen selection or interspecific hybridization of pigeonpea.
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Notes |
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1 To whom correspondence should be addressed. E-mail: jpfly{at}lycos.com
2 Present address: Directorate of Rice Research, Rajendranagar, Hyderabad 500 030, India.
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