Aug 12, 1998 - The African oil palm, Elaeis guineensis Jacq. (Arecaceae), is a native of Africa and Central-South America and is extensively cultivated in ...
Indian Journal of Experimental Biology, Vol. 37, February 1999, pp. 169-172
Polyethylene glycol enhances in vitro germination and tube growth of oil palm pollen R Tandon", TN Manoharab, B H M Nijalingappab & K R Shivannaa ' aDepartment of Botany, University of Delhi, Delhi 110007, India bDepartment of Botany, Bangalore University, Bangalore 560 056, India Received 12 August 1998; revised 14 October 1998
Sucrose or boric acid used by earlier workers for in vitro germination of oil palm pollen has not been satisfactory as germination is inconsistent, pollen tubes are short and show several abnormalities. Incorporation of polyethylene glycol (PEG 10,000) resulted in consistent and good germination, with long and normal pollen tubes. The best response was obtained on a medium containing sucrose (2.5%)+boric acid (100 ppm)+PEG (10%) in which pollen tubes were over 10 times longer than those in the medium without PEG. Use of PEG as a component of pollen germination medium for other species is suggested. The African oil palm, Elaeis guineensis Jacq. (Arecaceae), is a native of Africa and Central-South America and is extensively cultivated in Malaysia and Indonesia. Oil palm is the highest yielder (4-6 tonslha/yr) among the oil yielding crops. The Department of Biotechnology, Government of India has launched an oil palm demonstration project (1988-89) covering approximately 3000 ha in Andhra Pradesh, Karnataka and Maharashtra to study the feasibility of large-scale oil palm cultivation in the country. Oil palm is monoecious and produces male and female inflorescences in alternate cycles of variable duration I . Cross-pollination is brought about by wind and weevils. Studies carried out in Malaysia have shown that insufficient pollination is one of the important factors , which reduces the yield , particularly in plantations with younger palms. To overcome this problem, ass isted pollinations are carried out using stored poll en 1.2 Our preliminary studies on some plantations in Karnataka have also shown the necessity for ass isted pollin ati on under con ditions whi ch prolong the female phase for long periods. Thus storage of pollen is important not onl y to carry out assisted pollination to augment yield but also in oil palm breeding programmes2 . In vitro germi nation, a simp le and convenient test, is genera lly used to assess the viability of stored pollen grains] So far, only sucrose or boric acid 2•4 has been used as germination med ium for oi l palm pollen. However, in sucrose or boric acid med ium germinati on is genera lly erratic, with short pollen tubes, which show abnollnal ities such as forki ng, swe lling and rupturing of ti ps4 (our unpu blished results). Sucrose+boric acid+calciu m nitrate and min eral clements such as magnesium, potassium and copper have bcen used over the years to achieve better po ll en germination and tube grOlv1h in a range of specics1.5-8 In recent years po lyethylene glycol (PEG ) of various mo lecular weights has been used to improve po llcn gClln ination and tube groW1h in vitro in a num ber of speciesR- D ' Co rrespo nde nt author
The present work reports the effects of boron, calcium, potassium and magnesium in association with PEG to improve germination and tube growth in vitro of oil palm pollen, leading to formulation of a new medium, whi ch gives consistently good in vitro germination and long pollen tubes (> I 0 times that obtained in the sucrose+boric acid medium). Materials and Methods Pollen grains collected from plantations of Tenera hybrid located near Bhadrawati (Karnataka) during November 1996 and March 1998 were used for the experiments. Male spike lets with freshly dehisced anthers were gently tapped on a glazed paper sheet to release the pollen grains. They were allowed to air-dry for 30 min . The dri ed poll en from different trees were mixed, placed in airtight sc rew cap glass vials and were brought to Delhi under uncontrolled .temperature conditi ons by surface transport. It took 3-7 days from the day of co ll ecti on before the pollen sampl es we re used for germin ation studies at Delhi . Under natural co nditions oil palm pollen is reported to rem ain viabl e for an average period of one week2 The viability of po ll en sampl e used on the day of each experiment was assessed th rough flu oresce diacetate (FDA) test l 4 Stoc kin solution of FDA in aceto ne (2 mg/ml ) was added dropwise to 10% sucrose solut ion until the solut ion showed persistent turbidity. Poll en grains were mounted in a drop of thi s freshl y prepared FDA soluti on and obse rved under a Nikon flu oresce nce microscope (usin g fi lter block B-2A compri sin g excitati on filter 450-490 nm , di chromati c mi rror 5 10 nm and barrie r fi lter 520 nm )15 The bri ght ye llow ish flu orescin g grains we re scored as viable. In the first set of experim ent s the effects of min era l element s were stud ied with or without po lyethylene glycol 10,000 (Sigma) (Tablc I). On the basis of the results of the first experiment , the best medium compos iti on was se lected and optima l concentrations of PEG and sucrose were standardized .
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In vitro gennination was carried out using sitting drop cultures 15 (20 fll) at 28°C. Hydration of pollen in vapour phase (prehydration) has been shown to increase pollen 3 gennination in many species . Pollen grains were routinely pre hydrated before culture by spreading them on a dry slide and maintaining the slide in Petri plates lined with moist filter paper (RH>95%) for I hr. Two sitting drop cultures were raised for each treatment and each treatment was repeated at least for three times. From each replicate >200
pollen grains were scored for gennination and about 50 pollen tubes were measured for tube length using randomly selected microscope fields from both .the cultures. The data presented are the mean of three replicates . For studying callose deposition in pollen tubes, pollen suspensions were mounted in a drop of water soluble aniline blue (0 .005% in 0.05M Na2HP04) and observed under Nikon fluorescence microscope using filter block UV -2A (excitation filter 330380 nm, dichromatic mirror 400 nm and barrier filter 420 nm)15. Photomicroscopy was done using B/W IIford PAN 100 ASA film .
Table 1- Effects of mineral salts and PEG on pollen germination and pollen tube growth. [Culture period: 150 min. Viability of pollen sample used : 62%; ±SE] Composition of Medium Sucrose (10%) Sucrose (10%) . +Boric acid (100 ppm) Sucrose (10%) +Calcium nitrate (300 ppm) Sucrose (10%) +Boric acid (100 ppm) +Calcium nitrate (300 ppm) Sucrose (IO%)+BK salts [Boric acid (100 ppm) +Calcium nitrate (300 ppm)+Potassium nitrate (100 ppm) +Magnesium sulphate (200 ppm) Sucrose (2.5%) +PEG (10%) Sucrose (2 .5%) +Boric acid (100 ppm) +PEG (10%) Sucrose (2 .5%) +Calcium nitrate (300 ppm)+PEG (10%) Sucrose (2.5%) +Boric acid (100 ppm) +Calcium nitrate (300 ppm)+PEG (10%) Sucrose (2.5%)+BK salts +PEG (10%) Table 2 -
Germination (%)
Tube Length
5.3 ± 0.1 11.5 ± 0.1
48.0 ± 0.1 123 .3 ± 1.9
1.6 ± 0.1
48.5± 0.1
2.4 ± 0.07
64.0 ± 0.1
o
o
22.2 ± 0.1
116.7 ± 1.7
53 .0 ± 0.9
222.0:t 2.9
1.2 ± 0.1
42 .0 ± 1.5
16.7 ± 0.2
105 .2 j : 1.1
26.1 ± 0.7
176.6 ± 1.6
Results and Discussion The results of the first experiment are presented in Table I . The viability of the fresh pollen ranged from 80-86%; the viability of pollen sample used for this experiment had come down to 62%. Thus unlike earlier report 2, oil palm pollen show considerable loss of viability in 7 days under uncontrolled temperature conditions. The concentration of PEG and sucrose used in this experiment were based on earlier studies in other species ll •12 . Boric acid and calcium are known to be standard components of almost all the 3 pollen gennination media used so far . In oil palm pollien boric acid alone with sucrose induced some gennination but pollen tubes were short and showed many abnonnalities at the tip (Fig. I a-c). Most of the pollen tubes grown for 4 hr showed deposition of callose at the tIP, a featUlre characteristic of growth-arrested pollen tubes 16. Further, callose plugs along the pollen tubes, a feature usually B observed in actively growing pollen tubes , did not fOlm even when the cultures were maintained for 8 hI. Calcium alone or in combination with boric acid, inhibited pollen gennination. These results suggest that oil palm pollen are deficient in boron but not in calcium. lExogenous calcium is toxic probably because of the presence of a sufficient amount of endogenous calcium. The mineral salts of 5 Brewbaker and Kwack (BK) medium , which are 11\0st commonly used for pollen gennination of a number of species, did not favour gennination of oi l palm pollen. Addition of PEG to the medium remarkably improved both gennination and tube growth (Fig. Id,e). PEG could also substantially overcome the inhibitory effects of BK salts. The best response was obtained in the PEG medium containing sucrose and boric acid . In this medium, pollen
(11m)
Effect of different concentrations of PEG and sucrose in boric acid containing medium on pollen germination and pollen tu be growth [Culture period: 150 min. Viability of po \len grains used for experiment: 60.5%. Values are mean±SE] Sucrose
PEG
O. 2.5 5.0 10.0 15.0
5.0
2.5
0 G
TL
13 .2 ± 0.4 25 .1 ± 0.4 27.2 ± 0.4 33.5 ± 0.5 33. 1 ± 0.3
82.2 ± 0.9 194.6 ± 1.3 151.6 ± 1.2 206.0 ± 1.3 197.2 ± 1.0
.
G=germination (%), TL=tube length (11m).
10.0
G
TL
G
TL
G
TL
38.4 ± 0.4 33.6 ± 0.4 44.3 ± 1.0 52.3 ± 0.7 40.0 ± 0.5
I 79.{) ± 1.1 165.4 ± 1.0 228.1 ± 2.0 336.0 ± 1.9 183 .9 ± 1.2
26.3 ± 0.6 38.6 ± 0.5 39.5 ± 1.4 50.7 ± 1.1 38.6 ± 0.4
204.0 ± 1.1 210.3 ± 1.1 185.1 ± 0.5 224.8 ± 1.1 247.4 ± 1.3
23 .2 ± 0.3 27.0 ± 0.5 34.3 ± 0.6 39.4 ± 0.4 17.8 ± 0.5
123 .7 ± 0.7 131.3 ± 1.4 170.9 ± 0.8 144 .9 ± 0.7 120.6 ± 1.3
TANDON el al.: POLYETHYLENE GLYCOL ENHANCES POLLEN GERMINA nON tubes were long, narrow and smooth without any abnormalities. Pollen tubes showed regular callose plugs (Fig. Ie). There was a 5-fold increment in tube length in PEG medium as compared to the medium without PEG in 4 hr (Fig. 2). Pollen tube growth did not show any significant increase beyond 2 hr in the control medium, while in PEG medium the tube growth continued beyond 4 hr. By 8 hr pollen tubes measured over 1.2 mm in the PEG medium (Fig. 2). In the second set of experiments optimal concentrations of PEG and sucrose in the most responsive medium (i.e. sucrose+boric acid+PEG) were investigated (Table 2). Concentrations of sucrose above 2.5% were inhibitory. Maximum germination and tube growth were obtained in a medium containing 2.5% sucrose and 10% PEG. A further increase in the concentration of PEG decreased pollen germination as well ilS tube length. Thus the medium containing 2.5% sucrose+ 100 ppm b'oric acid+ I 0% PEG
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10,000 was found to be the most suitable medium for oil palm pollen. We have been using this medium routinely to study germination of fresh as well as stored oil palm pollen samples. The germination response is uniformly high. In this medium fresh pollen (within one day after colIection) gave up to 73.4% germination (carried out at Bangalore University). Lower percentage of germination recorded in pollen samples used in the present experiments was obviously due to loss of viability during transit from Bhadrawati to Delhi as confirmed through the FDA test. Apart from improving in vitro polIen germination and polIen tube growth, recently PEG has been shown to replace sucrose as an osmoticum for induction of microspore derived embryos 17. PEG is a relatively inert osmoticum and cannot enter the celIs'8. Although the mechanism of PEG action on polIen germination and pollen tube growth . has not been clearly understood, it seems to
Fig. 1 - Fluorescence micrographs of cultured oil palm pollen grains stained with aniline blue. (a-c) Pollen cultures in control mediwn (sucrose lOo/o+boric acid I 00 ppm) grown for 150 min (a), 4 hr (b) and 8 hr (c) Pollen tubes are short and show abnormalities in the fo~ of swollen and branched tips (b,c) and deposition of callose at the tip (arrow heads in c). (d,e) Pollen cultures in PEG medium (sucrose 2.5o/o+boric acid 100 ppm+PEG 10%) grown for 150 min (d) and 4 hr (~ . Germination is good and tubes are long and straight with deposition of regular callose plugs (arrow-heads in e) similar to those growing in the style.
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___ sucrose (10%) + boric acid (100 ppm)
120
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--
..• . . sucrose (2.5%) + boric acid (100 ppm) + PEG (10%)
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Time (h) Fig. 2 -
Kinetics of pollen tube growth in the control and PEG media.
provide a better osmoticum than sucrose. Sucrose readily enters the pollen grain and besides serving as a substrate for 8 respiration it also increases the internal osmoticum' which may not be conducive to pollen tube growth. PEG is also believed to regulate the permeability and stability of 8 plasmamembrane • The medium developed in the present investigation is much better than the earlier media used for oil palm pollen. Since PEG has be'!n shown to improve in vitro gernlination and more importantly tube length of a number of species of diverse families , it may be worthwhile to test the efficacy of PEG (of higher molecular weights) in the gernlination medium of other species particularly of refractory 'systems. The authors are grateful to Professor H Y Mohan Ram for useful discussions . This work was carried out under a research project sanctioned by the Department of Biotechnology, New Delhi.
References I Hartley C W S, The oil palm, II edition (Longman, London, New York) 1977. 2 Hardon J 1 & Davies M D, up Agric, 5 (1969) 59. 3 Shivanna K R & lohri B M , The angiosperm pollen: Structure andfunclion (Wiley Eastern, New Ddhi) 1985.
4 Rao A N & Eng L L, Curr Sci, 36 ( 1967) 641 . 5 Brewbaker J L & Kwack B H, Am J Bot, 54 ( 1963) 1069. 6 Roberts I N, Gaude T C, Harrod G & Dickin son H G , Theor Appl Genet, 65 (1983) 231 . 7 Hodgkin T, Cruciferae Newsletter, 8 (1983) 62. 8 Read S M, C larke A E & Bacic A, Protoplasma, 177 (1993) I.
9 Zhang H-Q & Croes A F, Act Bot Neerl, 3 1 (1982) 11 3. 10 Leduc N , Monnier M & Douglas G C. Sex Plant Reprod. 3 ( 1990) 228. II Shivanna K R & Sawhney V K, J up Bot, 46 (1995) 1771 . 12 Shivanna K R, Saxena N P & Seetharama N , IntI Chickpea and Piegonpea Ne wsletter, 4 ( 1997) 28. 13 Subbaiah C C, Can J Bot, 62 (1984) 24 73 . 14 Heslop-Harrison J & Hes lop-Harriso n Y, Sta in Technol, 45 (1970) 115 . 15 Shivanna K R & Rangaswamy N S, Pollen biology. A laboratory manual (Springer-Verlag, Berli n, Heidelberg, New York) 1992. 16 Heslop-H arri son J, Int Rev Cytol, 107 ( 1987) I. 17 Ilic-Grubor K, Attree S M & Fowke L C, Plant Ce ll Reports. 17 (1998) 329. 18 Powell 111 G M, in Handbo ok of waler soluble glllm and resins, edited by R E Davidson (McG raw-Hill, New Yo rk) 1980, 18.1-18.31.
