Pollen production and pollen viability in male jojoba plants

Pollen production and pollen viability in male jojoba plants

Industrial Crops and Products 18 (2003) 117 /123 www.elsevier.com/locate/indcrop Pollen production and pollen viability in male jojoba plants Yiftac...

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Industrial Crops and Products 18 (2003) 117 /123 www.elsevier.com/locate/indcrop

Pollen production and pollen viability in male jojoba plants Yiftach Vaknin a, David Mills b, Aliza Benzioni b,* a

b

Department of Pomology, 1 Shields Ave., University of California Davis, Davis, CA 95616, USA The Institutes for Applied Research, Ben-Gurion University of the Negev, PO Box 653, Beer-Sheva 84105, Israel Received 5 June 2002; accepted 26 March 2003

Abstract Jojoba (Simmondsia chinensis (Link) Schneider) is a dioecious wind pollinated shrub native to the Sonora desert of the southwestern USA and Northern Mexico. A comprehensive program for the selection of female cultivars was conducted in Israel for many years. However, selection for male cultivars as pollen sources has largely been neglected. The current use of male seedlings with their large variation in bloom period, amount of pollen produced, and pollen viability, could result in insufficient pollination and reduced yields. The study was conducted at Hazerim plantation, Israel (northern Negev desert), on 8 /9-year-old male jojoba plants. Pollen production was investigated 24 and 48 h after removal from the plants. Pollen viability was tested both in vitro and in vivo. Pollen production, in percent pollen from inflorescence fresh weight, was the highest during peak male bloom. The greatest amount of pollen having the best germinability was collected 24 h after the inflorescences were removed from the plants. In vitro germinability of jojoba pollen was affected by pollen source, and by sucrose concentration of the germination solution. Jojoba pollen from all pollen sources germinated intensively on the stigmas of the female flowers, but the percentage fruit set was different for various pollen donors. Several male plants from our study showed promising traits as potential male cultivars in terms of pollen production and viability. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Artificial pollination; Fruit set; Jojoba; Pollen germinability; Simmondsia chinensis (Link) Schneider

1. Introduction Jojoba (Simmondsia chinensis (Link) Schneider) produces a unique liquid wax commonly known as jojoba oil, and its derivatives have a wide range of potential uses. Today, its main use is in cosmetics and pharmaceuticals (US National Research

* Corresponding author. Tel.: /972-8-646-1970; fax: /9728-647-2984. E-mail address: [email protected] (A. Benzioni).

Council, 1985; Wisniak, 1987). A comprehensive program for the selection of female cultivars was conducted in Israel (Benzioni, 1995; Benzioni et al., 1999) of which several cultivars are now being regularly propagated for cultivation in new plantations. The bloom period of jojoba in Israel usually extends from February to March, but in some years, due to climatic fluctuations, it may begin in January and end as late as April. Out-ofseason bloom occurs in some cultivars in the autumn (Nerd and Benzioni, 1988; Benzioni et al., 1999). Thus, during a rather long period,

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several female cultivars are in bloom and require extended bloom periods of males for successful pollination. Selection for male cultivars as pollen sources, however, has largely been neglected. Currently in Israel, male jojoba plants originate from seeds and are grown along with female seedlings as a separate row for every 15 female rows (Benzioni and Ventura, 1998). The large variation in most bloom aspects of the male plants such as bloom period, amount of pollen produced and viability of the pollen, could result in insufficient overlap between pollen availability and stigma receptivity resulting in low fruit set and reduced yields. Poor fruit set and flower abortion are observed frequently in autumn out-of-season bloom and earlyto-bloom female cultivars such as ‘Q-106’, ‘Q-104’ and ‘Negev’ (Benzioni and Ventura, 1998; Benzioni et al., personal communication, 2002). One way to ensure pollen availability is to screen and select for male jojoba plants with desirable traits including production of large quantities of highly viable pollen, and bloom period at the same time as the female plants in the plantation. The objectives of this study were to investigate the various aspects of jojoba pollen production from several individually selected male seedlings and to establish techniques of pollen collection for future management of jojoba pollination.

copious amounts of pollen and a hermaphroditic plant (‘Hermaphrodite’) having fully developed anthers and partially developed ovaries and stigmas, both originally brought from Riverside, California. 2.2. Pollen production Pollen was collected before peak bloom and during peak bloom from male inflorescences with dehiscent anthers. Inflorescences (about 200 /700 g) were picked, weighed, and then spread over paper sheets in a ‘pollen room’ (23 /25 8C and 35 and 45% RH) for 24 h (first collection). Pollen shed overnight was cleaned by passing it through a 100 mm mesh sieve, and weighed. Some of the 24 h old inflorescences were subjected to the same procedure for an additional 24 h (second collection). Pollen from males 25, 26, and 29 was stored for 10 days over silica gel desiccant at /18 8C. Pollen from males ‘Rav-On’ and ‘Hermaphrodite’ was stored for 1 day before it was used for in vivo germination. Some of the collected ‘Rav-On’ inflorescences were pre-washed by dipping them in distilled water and excess water was shaken off before pollen collection was continued. Fresh pollen from both pre-washed and unwashed inflorescences was tested for in vitro germinability and was then kept desiccated at /18 8C. 2.3. Pollen germination

2. Materials and methods 2.1. Plant material Pollen was collected from 8 to 9-year-old male jojoba plants in the Hazerim plantation (Northern Negev, Israel) (Benzioni et al., 1999). Over 30 male seedlings were observed and analyzed at the seedling population in Hazerim plantation and three male plants were selected for more detailed studies (‘25’, ‘26’ and ‘29’) based on bloom dates, morphological characteristics largely related to the density of inflorescences and plant size, and on effect on wax composition (for more details see Benzioni and Vaknin, 2002). Two other unique plants were studied; ‘Rav-On’ a male with unusually big and branched inflorescences producing

2.3.1. Germination in vitro Pollen was pre-hydrated before germination in humidified Petri dishes for 90 min (at this time pollen water content was increased by 30/35%). Pre-hydrated pollen was incubated in 0.2 ml aqueous solution containing 6 /20% (w/w) sucrose, 160 mg l 1 boric acid and 240 mg l 1 calcium nitrate tetra hydrate, in micro wells for 20 /24 h under room conditions (23 /25 8C and 35 /45% RH). Germination percentages were determined with an inverted microscope for 100 grains in each of six wells. Pollen was considered to have germinated when the pollen tube was at least as long as the diameter of the pollen grain. Based on the results of pollen germination in different sucrose concentrations (Fig. 1), it was decided

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Fig. 1. Pollen germination from three different jojoba male plants as affected by various sucrose concentrations of the germination solution. Squares, male ‘25’; circles, male ‘26’; and upward triangles, male ‘29’. Values are mean/S.E.

that all further germination tests be conducted with 15% sucrose solution.

2.3.2. Germination in vivo 18 3-year-old female plants were used. About 30 /32 female flowers were pollinated with pollen from each male pollen donor. Pollen from males ‘25’, ‘26’ and ‘29’ was stored for 10 days under desiccation at /18 8C and pollen from males ‘Rav-On’ and ‘Hermaphrodite’ was stored for 1 day, prior to pollinating the flowers. Pollen was applied to stigmas of female flowers (cv ‘Benzioni’) in a greenhouse by dipping the stigma in pollen placed on a small spatula. After 7 days, the stigmas were removed and preserved in Histochoice Tissue Fixative (aMResco, Solon) and kept refrigerated for later study of pollen germinability on the stigma using a modification of the aniline blue epifluorescence method (Martin, 1959). Fruit set of the pollinated flowers was scored 2 months later. Preliminary tests showed that stigma removal more than 4 days after pollination did not interfere with fruit set.

2.4. Statistical analysis STATVIEW 5.0 (SAS Institute Inc.) software was used for data analysis. The relationship between bloom date (Julian dates, days from 1 January 2000) and percentage of pollen collected from green weight was analyzed by Pearson correlation coefficient. Two-tailed t -tests were used to analyze data on the following: (a) percentage of pollen grains from fresh inflorescences in first and second collections; (b) the total weight of pollen in percentage from fresh weight collected before and during peak bloom; (c) percentage means of in vitro germinability of pollen from first and second collections; and (d) in vitro germinability of fresh pollen from pre-washed and unwashed ‘Rav-On’ inflorescences. All data, except for means, were transformed prior to statistical analysis. Arcsine transformation was applied on percentages from fresh weight of collected pollen and on percentages of in vitro germinability. Data are represented as mean9/ standard error (S.E.). Values are reported as significantly different when P was 5/0.05.

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3. Results

3.1. Pollen production Pollen production (percentage from inflorescence fresh weight) increased linearly as the bloom period progressed during February and March 2000 (Fig. 2; n/48, r /0.51, P /0.0002). When male inflorescences were harvested prior to peak bloom, the second collection from the same inflorescences had significantly higher production of pollen compared with the first collection (second /first) (t6 //2.78, P /0.032) (Table 1). Conversely, when male inflorescences were collected during peak bloom, the first collection had significantly higher production of pollen compared with the second collection (first /second) (t6 / 6.34, P /0.0007) (Table 1). Total pollen production prior to and during peak bloom was similar with a slightly non-significant increase at peak bloom (t6 /2.06, P /0.08) (Table 1).

3.2. Pollen germinability in vitro during bloom season Pollen germinability for first collection from the same inflorescences was significantly higher than that for the second one during peak bloom (69.1%9/11.1 and 26.5%9/5.7, respectively; t14 / 3.424, P /0.004). Similar results were observed prior to peak bloom. Pollen germinability of male plants was highly variable. Nevertheless, a definite pattern was noted in germinability of pollen from various male plants, collected throughout the bloom period: in early February, germination values showed very high variation, from as low as 8 /69% and from mid-February germinability ranged between 21 and 95% (Fig. 3). In late February to early March, variation in germination rates significantly decreased and values stabilized in the mid to high range; 43/73% in 21 February 2000 and 29 February 2000, and 52/68% in 6 March 2000 (Fig. 3). Pre-washing of the ‘Rav-On’ inflorescences prior to pollen collection significantly

Fig. 2. Amount of pollen harvested at different dates. Dates are expressed as Julian days; beginning 1 January 2000. Pollen yield is expressed as percentage of pollen fresh weight (collected after 24 h in a pollen room) from inflorescences’ fresh weight at day of harvest. y/0.034X/0.106, r/0.51, P /0.0002.

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Table 1 Fresh weight of pollen per fresh weight of inflorescences collected from four jojoba male plants before peak bloom (2 February 2000) and during peak bloom (7 February 2000) Harvest date

2 February 2000 7 February 2000

Pollen fresh weight (% of inflorescence fresh weight) First collection

Second collection

Both collections

0.869/0.16 2.489/0.26

1.339/0.06 0.539/0.17

2.199/0.14 3.019/0.37

The weight of inflorescences harvested from each male plant at each date ranged from 200 to 670 g.

increased pollen germinability from 29.3%9/1.56 to 66.8%9/1.60 (t10 //16.05, P B/0.0001).

3.3. Pollen germinability in vivo The number of pollen tubes observed on the stigma was extremely high for all pollen sources and accurate counting was impossible. Fruit set of the pollinated flowers ranged from about 47 to 87% depending on the male pollen donor (Table 2).

4. Discussion The yield of pollen collected, i.e. amount of pollen per fresh weight of harvested inflorescences, was affected by pollen source and bloom date. However, the linear increase in the amount of pollen collected per inflorescense with bloom date (Fig. 2) explains only about 26% (r2 /0.26) of the total variation in yield. Other factors such as genetic variability and level of progress in bloom of the individual male may be involved. During peak bloom, more flowers are dehiscing or about to dehisce. Therefore, the majority of

Fig. 3. Pollen germination from different jojoba male plants during the 2000 bloom period. Date is expressed as Julian days (beginning 1 January 2000). Values are mean9/S.E.

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Table 2 Fruit set of hand pollinated jojoba female flowers from one female cultivar (‘Benzioni’) with pollen from five male pollen donors Male donor

‘25’

Collection date Pollination date

20 February 2000 ‘26’ 20 February 2000 ‘29’ 20 February 2000 ‘Hermaphrodite’ 6 March 2000 ‘Rav-On’ 6 March 2000 ‘Rav-On’ fresh 14 March 2000

Fruit set (%)

2 March 2000

83.3

2 March 2000

64.0

2 March 2000

46.7

7 March 2000 7 March 2000 14 March 2000

58.1 50.0 87.1

‘Rav-On’ was used after storage and without storage-fresh. N/30 /32.

pollen are shed within the first 24 h after collection from the plant (first collection). However, prior to peak bloom, many flowers may not have reached maturity, and therefore, more time was required for the anthers to dehisce and shed the majority of pollen (between 24 and 48 h). The total weight in percentage from fresh weight of pollen harvested prior to and during peak bloom was similar. However, the proportion of pollen harvested during first and second collections from each inflorescence was different (second /first before peak bloom and first /second during peak bloom; Table 1). In terms of germinability, during peak bloom, the pollen shed within the first 24 h was of much higher quality than those shed between 24 and 48 h. Because efficiency of pollen collection corresponds to the bloom phenology of the individual plants, collecting pollen from a specific plant near its peak bloom could both increase the efficiency of the collection by acquiring the major proportion of pollen within the first 24 h and maintain a higher pollen quality. Buchmann (1987) reported that certain wild and cultivated male jojoba plants produced pollen with germinability ranging between 30 and 60%. He suggested that genetic or environmental causes may affect germinability. Our test on pollen germinability of male jojoba plants growing under

the same environmental conditions showed significant differences among those males, which suggests a genetic source as an important factor (Fig. 3). The large variation in pollen germinability, especially in early blooming plants (Fig. 3), emphasizes the need to select for males with high quality of pollen for fertilization of early-, midand late-blooming females. Tests of germinability of pollen from a single plant ‘Rav-On’ revealed an increase in germinability after washing the inflorescences. We suggest that washing the inflorescences will remove dust and dirt particles that accumulate on the flowers under field conditions. The plants grow under desert conditions with mild to strong sand storms that create a dense cover over the entire plant and may very well be detrimental to pollen germination. We also suggest that by pre-washing the inflorescences pollen is removed from the already dehiscent anthers. This pollen had already been exposed to ambient conditions and may have lost some viability. Fertilization of female flowers with pollen from several pollen donors resulted in relatively high fruit set (Table 2). The differences between the pollen donors may have been affected by the pollen source, and by reduction in viability due to short-term storage (e.g. ‘Rav-On’ and ‘Rav-On’ fresh; Lee et al., 1985; Barnabas and Kovacs, 1997). The results also suggest that some male plants such as ‘Rav-On’ ‘25’ and ‘26’ may be good pollen sources for both new mixed-sex plantations and for artificial pollination of existing plantations or new all female ones. In conclusion, screening for superior male jojoba plants with large pollen production and high quality pollen, as potential pollen donors in future jojoba plantations was partially achieved, but additional research on other male plants and tests on propagated plants used in this study should be implemented.

Acknowledgements We thank Shoshana Avni and Galina Agafanova for their invaluable work in the laboratory, and to Katherine Pinney for editing the manuscript.

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Buchmann, S.L., 1987. Floral biology of jojoba (Simmondsia chinensis ) an anemophilous plant. Desert Plants 8, 111 /124. Lee, C.W., Thomas, J.C., Buchmann, S.L., 1985. Factors affecting in vitro germination and storage of jojoba pollen. J. Am. Soc. Hort. Sci. 110, 671 /676. Martin, F.W., 1959. Staining and observing pollen tubes in the style by means of fluorescence. Stain Technol. 34, 125 /128. Nerd, A., Benzioni, A., 1988. Effect of water status, genetic background gender and fertilizer on flowering in jojoba. Adv. Hort. Sci. 2, 48 /51. US National Research Council, 1985. In: Jojoba: New Crop for Arid Lands, New Raw Material for Industry. National Academy Press, Washington, DC, Library of Congress Catalog number 85-061501. Wisniak, J., 1987. The Chemistry and Technology of Jojoba Oil. American Oil Chemical Society, Champaign, IL, pp. 1 / 73.