Pollen Germination in vitro Pollen Germination in vitro

Pollen germination in vitro is a reliable method to test the pollen viability. It also addresses many basic questions in sexual reproduction and particularly useful in wide hybridization. Many pollen germination medium ranging from simple sugars to complex one having vitamins, growth regulators, etc. in addition to various minerals have been standardized to germinate pollen artificially. The different media, successful pollen ger - mination methods, procedures from pollen germination studies with wheat, rye, brinjal, pigeonpea and its wild relatives are discussed.


Introduction
Pollen germination in the stigmatic tissue of Portulaca was first observed as early as 1824 by Amici and later he observed the germinating pollen tube entering ovule. Pollen acts as a vehicle for the transfer of male gametes to embryo of female plant. Pollen viability is an important factor in successful hybridization which may last from few minutes mostly in selfpollinated crops to many hours or days in cross pollinated crops. Assessing pollen viability is very crucial in artificial pollination especially involving different species or genera. Among diverse techniques used to assess pollen viability, in vitro pollen germination is the most reliable method. Pollen has been germinated in variety of media which differs from species to species and even for different varieties of a crop [1,2]. Linskens [3] used simple sucrose/boric acid media and later many complex medium have been reported with addition of polyethylene glycol, various amino acids, etc. [4][5][6]. Among the many PGMs the one developed by Brewbaker and Kwack [7] has been widely used with some alterations.
A brief discussion was made in this chapter to develop a pollen germination protocol for a crop/species.

Pollen germination medium
The composition of some of the successful media used pollen germination are given below Brewbaker and Kwack medium [7] • 10% sucrose PEG medium [4] • 0.1 to 1.1 M PEG 400, • 100 mg l −1 Boric acid EACA medium [1] Raffinose medium [9]. Tryptone PGM of rye [10] Agarose medium [11]: A basic medium contains a sugar, calcium nitrate and boric acid to which poly ethylene glycol, vitamins, amino acids, growth regulators etc. are added to make a complete pollen germination medium. The pH and temperatures are also important factors.

Pollen germination methods
Pollen is collected from freshly opened flowers and enough pollen is dusted over medium and culture by any one of the following methods: • Cavity slide technique (Rangaswamy and Shivanna [12,13]) : It is used for liquid pollen germination medium(PGM). A drop of medium is placed in the cavity, pollen is dusted over and it is covered with a dust free cover slip with its periphery sealed with Vaseline. It creates a required relative humidity inside. The slide is placed inversely over a pair of glass • Agarified medium in Petri dishes [14]: Pollen was extracted from fresh buds (with bud break) and was spread evenly on a drop of medium in a Petri dish and covered with Petri plate lined with moist filter paper. The plats were incubated in a BOD incubator with a temperature of 18°C/20.5°C depending on the pollen sample • PGM droplet technique [15]: In a Petri dish a droplet of pollen germination medium was placed using a glass rod. Thus drops of different media could be placed within few centimeter distance between them For example nine media can be placed in 3 × 3 fashion (Figure 2).
Further in order to have a better visualization, a drop of stain was placed carefully over the medium droplet before observation. The extra stain was removed with a piece of dried filter paper. (Acetocarmine, and components of Alexander stain viz., Malachite green, Orange G, Aniline Blue and acid fuchsine may be used).
• The cellulose membrane/agarose culture apparatus [11]: Rectangular agarose pad was prepared over a microscopic slide with PGM containing 0.5% agarose. Appropriate size of cellulose membrane was cut and layered over agarose pad. The pollen is placed over the membrane and cultured in a moist chamber. In our experiments initially liquid medium was used but profuse pollen bursting was seen due to quick hydration. To overcome this problem later agarified media were used by adding 0.5 to 1% agar to pollen germination medium. PGM droplet technique is a further refined technique which may be used effectively to fix the initial levels of inorganic salts level for factorial experiments. Each season, at least 200-300 fresh media were prepared and tested. This allowed large number of media to be tested in a single day, thus avoiding preparation of fresh medium each time and furthermore, more than two genotypes or species could be tested simultaneously.

Effect of different components of pollen germination medium (PGM) on pollen germination
Sugars: The sugar in the medium acts as an osmotic regulant which regulates the diffusion rate of water from the medium into pollen grains [16] and sucrose is generally used in PGM. The failure of pollen to germinate and its bursting may indicate lack of sugars within pollen grains and critical dependence on external supply. Sucrose has given satisfactory pollen germination for pigeonpea, wild pigeonpeas and wild brinjals whereas it was a poor osmatic regulant for pollen of wheat, rye and brinjal. Maltose was used as carbon source for these species.
Boron and calcium nitrate: The mineral requirements are also different for any two species. Pollen grains are believed to be deficient in boron, which is normally compensated by high levels of boron present in stigma and style. Boron combines with sugar to form a sugar-borate complex which facilitates translocation of sugar molecules. Boron is reported to be toxic to plants even at as low as 5.1 ppm [17]. However, pollen seems to tolerate very high concentration of boron. Visser [18] showed that certain species of crop plants require as much as 1200 ppm boric acid for optimal germination and tube growth. Boron deficiency leads to pollen tube bursting as its required in the pollen wall structure [19]. Calcium is involved in cationic balance and is essential for tube elongation [7]. Pollen germination involves many ions with Ca 2+ as the key player and extracellular calcium proved essential for pollen tip growth [20]. It was observed that the grass pollen (wheat and rye) requires very low levels of these minerals (30-50 mg l −1 ) as compared to the dicots (pigeonpea and brinjal) (300-400 mg l −1 ). Pollen Germination in vitro http://dx.doi.org/10.5772/intechopen.75360

Polyethylene glycol (PEG):
It is known to be a non-penetrating osmotic agent that decreases water potential of culture medium [21]. In pollen grains, PEG is considered to regulate the permeability of plasma membrane and to give stability to the pollen tube membrane [6] and to give stability to the pollen tube membrane. PEG of different molecular weight has been in pollen cultures of different species and in all cases it has a promontory effect [4,6,22]. Generally 10-15% of PEG was added to PGM. PEG 4000 was preferred for pigeonpea and brinjal whereas wheat and rye pollen germination satisfactorily with addition of PEG 6000.
Peptone: Inclusion of peptone arrested pollen tip bursting in wheat at concentration 80-120 mg l −1 which was used in PGM for the first time [2]. Peptone has been used in plant tissue culture for various effects such as induction of shoot regeneration in avocado [23], promotion of hairy root formation in ginseng [24] etc. Addition of peptone, contained pollen tube burst to a great extent, it was less than 20% as compared to >85% in the medium which is devoid of peptone however, the level of peptone varied with genotypes.

ε-Amino caproic acid (EACA):
It is an amino acid derivative, a saturated six carbon fatty acid (C 6 H 12 O 2 , CH 3 (CH 2 ) 4 -COOH) which occurs in milk fats. This immunosuppressor has been used to overcome self-incompatibility and incongruity in Phaseolus [25] and Vigna [26,27]. It has been used as a component of PGM of pigeonpea for the first time [1] and subsequently in the medium of wheat and rye [2,10].
Tryptone: It was good growth-stimulating nitrogen sources used in the cultivation of Trichoderma hamatum and T. harzianum [28]. It was found to be the best organic nitrogen source for kefiran production by Lactobacillus kefiranofaciens (Dainiel et al., 2015), asparaginase production from Enterobacter cloacae [29], production of biosurfactant by Bacillus subtilis [30]. In rye pollen germination, tryptone at 50-75 mg concentration gave satisfactory and reproducible level of pollen germination [2].
Besides many organic supplements were added to the PGM to enhance smooth pollen tube growth such as casein hydrolysate, vitamins etc.

A protocol to standardize in vitro pollen germination medium
To standardize pollen germination medium for any crop initially a set of media are used. Based on the response, one of the media is picked up and modified. Here a procedure to develop a PGM for pigeonpea and its wild species is discussed. Initially two sets of key media(PGM) were used Set I consisted of Brewbaker and Kwack (BK) medium [7] with 1% agar at different levels of sucrose viz., 10, 20, 30 and 40% (A-D). With the addition of polyethylene glycol (PEG) 4000 at 15% concentration to each of A, B, C and D; the media E, F, G and H, respectively, were obtained. These were designated as Set II.

Composition of Brewbaker and Kwack's (BK) medium
Key media Based on the germination/initiation of germination and bursting of pollen, one of the key media was first selected (Figure 3). The complete medium for each species was then standardized by altering the concentration of sucrose, boric acid and/or calcium nitrate one by one to obtain maximum pollen germination and good pollen tube growth. First sucrose concentration and temperature were varied to prevent bursting of pollen before germination. Secondly, varying concentrations of boric acid was tried viz., 50, 100, … 300 ppm keeping 300 ppm of calcium nitrate (as in BK medium) and other components unchanged. Lastly, the optimum concentration of calcium nitrate was determined (100, 200, … 600 ppm). The observations were recoded for three best concentrations of each boric acid and calcium nitrate.

Standardization of in vitro pollen germination medium for C. platycarpus
In the medium (B) containing 20% sucrose, 1% agar and standard Brewbaker and Kwack medium's salts, pollen of C. platycarpus showed 99.15% germination and tube length over 172 μm in 1 h duration at 20.5°C. Lowering the concentration of sucrose to 10% (medium A) and leaving other constituents unchanged gave 61.60% germination with a mean tube length of over 83.88 μm. Though the medium (C) containing 30% sucrose gave 94.25% germination, the tube length was reduced considerably (33 μm), also pollen tube burst at the tips.
The best medium which gave 99.15% germination for in vitro pollen germination and pollen tube growth of C. platycarpus was Sucrose 10% Boric acid 100 mg l −1

Standardization of in vitro pollen germination medium for C. volubilis
On key media G (from Set II) the pollen of C. volubilis showed initiation of germination (budding) but almost all pollen grain showed bursting before or after budding. To contain bursting of pollen, sucrose level was increased to 35% and also the temperature was reduced to 18°C.
In order to improve germination, first boric acid concentration was altered keeping Ca(NO 3 ) 2 at 300 ppm. At 250 ppm, maximum pollen germination was obtained (approximately 63%) and increasing or decreasing boric acid concentration over 250 ppm did not improve germination.
Then This gives 94.26% pollen germination.

Standardization of in vitro pollen germination media for C. cajan vars. Pusa 33 and IMS-1
In the medium D (Set I) only initiation of germination was observed whereas no germination was observed in media A, B and C. On varying the concentrations of boric acid and calcium nitrate, it was observed that the medium with 40% sucrose, 250 ppm H 3 BO 3 , 300 ppm Ca (NO 3 ) 2 , 1% agar and BK salts gave 43.80% germination with abnormal pollen tubes.
From Set II, medium G showed over 45% germination (Figure 2) but there was a lot of bursting of pollen grains; medium F also showed little germination. To control the bursting of pollen grains sucrose level was raised to 37.5% and further, different combinations of boric acid and calcium nitrate were tried. Even though over 87% germination was seen, almost all the pollen grains showed bursting after germination. Among the permutations and combinations of media tried by changing the concentration of boric acid and calcium nitrate, six combinations showed better germination (70-80%) but accompanied with bursting of pollen tubes.

Pretreatments
In order to standardize optimal media for pollen germination different methods of preconditioning the pollen were attempted. Heat treatment of pollen at 40°C for 30 min or at 60°C for 20 min did not improve germination of C. cajan pollen. Pre-hydration for 30 min in Petri dish lined with moist filter paper also did not improve germination. The stamen of pigeonpea is diadelphous (five short and five long stamens). Pollen extracted separately from short and long stamens showed approximately 70% germination. Pollen extracted from single anthers also showed about 70% germination. Thus, germination percentage did not vary between different anthers. Out of six combinations of media tried, the PGM with 250 ppm boric acid and 300 ppm calcium nitrate showed maximum normal pollen tubes.
Another pre-treatment of pollen was tried by incubating young buds (12 h or 36 h before anthesis) at 20.5°C in (a) Petri dish lined with moist filter paper for 36 h.  Then upon anther dehiscence, pollen was collected and germination was tested on six different media. The pollen from treatments (a), (b) and (c) showed good germination on medium G 5 but burst soon after.
For treatments (d) and (e) media G 1 -G 6 were used both for pre-treatments and germination tests. The pollen from the treatment (d) showed much variation for germination and bursting.
Pollen extracted from the treatment (e) showed improved germination for C. cajan var. Pusa 33 and IMS-1. Among the media tested (G 1 to G 6 ) the medium (G 5 ) with 250 ppm H 3 BO 3 and 300 ppm Ca(NO 3 ) 2 showed over 92% germination for both varieties. Besides media G 3 , G 4 and G 6 also showed over 90% germination but there was pollen tube bursting. The result indicated pre-treatment of young buds (36 h before anthesis) in PGM (G 5 ) for 36 h and germinating pollen in the same PGM gave over 92% germination Ca(NO 3 ) 2 some other media (G 3 , G 4 and G 6 ) also showed over 90% pollen germination but there was bursting of pollen tubes at tips and variation in pollen tube length.
Thus, the complete media for both Pusa-33 and IMS-1 should have the following composition: To the above medium EACA at a concentration of 250-750 mg l −1 was added to arrest pollen tip bursting.
The results of studies on in vitro pollen germination of Cajanus cajan and its wild species are summarized in Table 1.
Based on the initial tests on C. lineatus it was found that modification of medium "G" could give optimum germination but due to non-availability of pollen, PGM could not be optimized.

Varietal response to PGMs
It was noticed that there are genotypic differences for pollen germination. In pigeonpea, among the genotypes tested, IDTSP51 alone did not require EACA, but for others the requirement varied(250-750 mg l −1 ). Similar response was seen with wheat pollen germination. The medium supporting more than 90% pollen germination were selected for each genotype though some of them showed pollen tube bursting which indicated the need for fine tuning of the medium. The genotypes HW 971 and HW 741 responded well in M19 giving >95% pollen germination and pollen tube length of >400 μm. This is adjudged as the best medium since it did not require supplementing either EACA or peptone. It is noted that some genotypes (HD 2833, HW 971, HW 1095 etc.) require EACA alone in PGM whose response were at par with medium having EACA + peptone. Some varieties such as MACS 6195, HW 1095 etc. showed better responses (>92% germination) in PGM with a combination of EACA (500 or 750 mg) and peptone water (100 mg). These varieties responded poorly when peptone water was increased beyond 100 mg which reduced the pollen tube length (

Conclusion
A simple or a complex medium may be developed to suit the germination requirement of pollen (binucleate or trinucleate pollen). The protocol suggested would give a better guidance in development of PGM. The author has developed pollen germination medium (PGM) for many crops viz. pigeonpea and its wild species, wheat, rye and brinjal by using the base constituents of PGM of Brewbaker and Kwack [7]. These media were supplemented with polyethylene glycol, e-amino caproic acid, peptone etc. besides the addition of BK salts. Grasses pollen so far considered as recalcitrant can also be germinated in the artificial medium e.g. wheat and rye. The grass pollen is released at high moisture content (30-40%) as compared to 1-5% in the case of orthodox species. This trait makes it unsuitable for in vitro germination. With combination of maltose, PEG and tryptone/peptone contained the initial pollen bursting and resulted in successful pollen germination of wheat and rye. The grasses pollen was so far classified as recalcitrant [31]. It is observed that monocots require low salt content than the dicots in the pollen germination medium. Also wild species require minimal medium than the domesticate ones.