Influences of Cross Pollination on Pollen Tube Growth and Fruit Set in Zuili Plums (Prunus salicina)

Zuili is a cultivar of plum (Prunus salicina L.) that is distributed in the Jiaxin district in Zhejiang Province, southern China. It has been planted for over 2000 years, probably because of its high fruit quality with abundant juiciness, high sugar (higher than 17%), low acidity (less than 0.8%) and its high acceptance by most consumers (Chen et al. 1981). The fruits are globular with a faint suture with red skin and yellow flesh, and covered with a thin powder. Despite the high quality of the fruit, cultivation of this plum in this area has been limited, mainly due to inadequate fruit set (Chen et al. 1982). Self-incompatibility might be one reason for the poor fruit set in Zuili. The trees produce large numbers of flowers but the fruit set percentage is usually very low, which has discouraged growers to cultivate this plum. There are few studies which have clearly confirmed the causes of the poor fruit set. This lack of cultural information is inhibiting the industrial development of Zuili production.

It is well established that fruit set and yield are strongly dependent on genotype and genotype interactions for many important fruit crops within the family of Rosaceae, such as apples, pears, cherries, apricots and almonds. Insufficient fruit set in plums may be due to a genetic predisposition for abnormal embryo sac development (Thompson and Liu 1973), and low temperature conditions during and after bloom time that result in poor pollen tube growth (Jaumien 1968). Poor fruit set also results from a shortened effective pollination period of the stigma as well as discrepancy between ovule longevity and the time required for the pollen tube to reach the egg cell (Williams 1965, 1970).

Improved and sustainable production of Zuili plums would be possible by interplanting proper pollinizer plants if cross-compatibility between Zuili is elucidated. In this study, we focused on the compatibility of several pollen parents for Zuili by studying pollen germinability and tube growth in vitro, pollen tube growth into the pistil, and ovule development. Finally, we investigated the effect of cross- and self-pollination on the fruit set of Zuili.

Results

Quantity and quality of pollen grains

As shown in Table 1, different results were obtained for pollen viability when assessed by in vitro tests and fluorescent microscopic examination of the Zuili stigma surface (Figure 1). Among the four cultivars, the highest pollen germinability, a notably high value of over 37%, was recorded in Black Amber pollen, about 25% in Miili and Hongxinli, while only 13.2% in Zuili pollen. However, tube length and rate of tube growth were more preponderant in Miili pollen than those in other cultivars.

Table 1. Pollen germination and pollen tube length after 6 h of in vitro cultivar^a

Cultivar	Germination (%)	Tube length (mm)	Rate of tube growth (μm/min)
Zuili	13.0cb	0.5c	1.4b
Black Amber	37.7a	0.8b	2.3a
Hongxinli	25.8b	0.4c	1.2b
Miili	27.6b	1.1a	3.0a

• ^aPollen grains were incubated on agar 1%+ sucrose 15% medium at 25 °C.
• ^bValues with common alphabets in each column are not significantly different at 5% level by Duncan's multiple range test.

Black Amber pollen grains germinated well also on the Zuili stigma surface (Figure 1A). On the contrary, Zuili pollen, the self pollen, exhibited significantly poorer germination than other pollen sources. The most pollen tube tips swelled or were arrested to grow further (Figure 1B). A notable number of pollen grains were found on open-pollinated stigmas but pollen tubes that penetrated into the style were few (Figure 1C).

Pollen tubes, elongated into various parts of a pistil, were easily distinguishable by fluorescent microscopic observation of cross sections stained with aniline blue (Figure 2). The most rapid pollen tube growth into the style base and upper ovary was observed in pistils cross-pollinated by Miili pollen when compared 4 d after pollination (DAP) (Table 2). The trend was also found at 8 DAP, although similar pollen tube growth was also detected in pistils pollinated by Hongxinli pollen. In pistils of self- and open-pollination treatment, few pollen tubes grew into the ovary even at 8 DAP. In pistils of self-pollination, pollen tubes grew slowly and only a small number of pollen tubes finally reached the micropyle through the obturator. A similar rate of pollen tube growth was detected for open-pollination treatment; the average number of pollen tubes reaching the micropyle was below 0.1. The number of pollen tubes that finally penetrated into ovule was higher in pistils pollinated by Black Amber and Miili pollen than by Hongxinli pollen. The numbers of pollen tubes counted in various parts of ovaries at 8 DAP decreased when measured at 14 DAP in each pollination treatment.

Table 2. Effect of cross pollination on pollen tube growth into various parts of Zuili pistils

Type of pollination and pistil stagea	No. of pistils examined	No. of pollen tubes per pistil
		Style base	Upper ovary	Locule top	Obturator	Micropyle
4 DAP
× Miili	16	1.5	0.7	0.2	0.1	0.0
× Hongxinli	15	0.5	0.3	0.1	0.1	0.0
× Black Amber	15	0.6	0.5	0.4	0.1	0.0
Open pollination	16	0.3	0.3	0.3	0.3	0.0
Self pollination	17	0.0	0.0	0.0	0.0	0.0
8 DAP
× Miili	27 (1)b	2.5	1.8	1.4	0.9	0.5
× Hongxinli	27 (2)	2.5	1.7	1.2	0.7	0.3
× Black Amber	15 (3)	1.0	0.9	0.6	0.6	0.5
Open pollination	17 (2)	0.4	0.3	0.3	0.2	0.1
Self pollination	23 (3)	0.4	0.4	0.2	0.1	0.0
14 DAP
× Miili	20 (2)	0.6	0.5	0.3	0.3	0.3
× Hongxinli	20 (1)	0.7	0.6	0.4	0.3	0.3
× Black Amber	15 (2)	1.1	0.7	0.5	0.5	0.5
Open pollination	16 (2)	0.3	0.2	0.2	0.1	0.1
Self pollination	17 (1)	0.4	0.2	0.1	0.1	0.0

• ^aPollination was carried out on April 3, and pistils were sampled 4, 8, and 14 d after pollination (DAP).
• ^bNo. of double pistils.

Ovule and embryo sac development

Zuili plum normally contains two ovules per pistil: the primary ovule within which an embryo sac develops and an abortive secondary ovule which normally degenerates. In some cases both primary and secondary ovules degenerate after pollination, in others both ovules remain viable (Figure 3). It is noted that there were two types of pistils in Zuili cultivar, single and double (Figure 4). Out of 50 pistils examined, 14 pistils were the double type, in which more than 90% of the ovaries were not viable. Degeneration of ovules in single type pistils was observed at 29.7% of the 74 ovules examined and 45.9% of the ovules contained no embryo sac (Table 4).

Table 4. Ovule development in normal and double Zuili pistils at the petal fall stage^a

Type of pistilb	Normally developed	Without embryo sac	Degenerated	Total no. of ovules
Normal	18 (24.4)c	34 (45.9)	22 (29.7)	74
Double	5 (8.9)	23 (41.1)	28 (50.0)	56
Total	23	57	50	130

• ^aP < 0.005, χ²= 10.3711.
• ^bRefer to Figure 1.
• ^cType of ovules (distribution %).

Discussion

There was significant inosculate between the pollinations as measured by both pollen tube growth and final fruit set. In vitro tests of pollen germination and tube elongation revealed that Black Amber pollen had the highest germinability and that Black Amber and Miili pollen tubes could elongate most rapidly. Such high abilities of pollen germinability and tube growth were also detected in pollen tube penetration into pistils. In the pistil pollinated with Hongxinli and Miili pollen, lager numbers of pollen tubes were counted in the style base, upper ovary, and locule top than in those pollinated by Black Amber pollen when examined 8 d after pollination. However, the number of pollen tubes decreased significantly at the obturator and micropyle in pistils pollinated with Hongxinli and Miili pollen, while in pistils pollinated with Black Amber pollen the decrease was smaller. This fact might indicate the higher compatibility of Black Amber pollen with Zuili pistils compared to other pollen sources. The highest fruit set percentage obtained from pollination using Black Amber pollen also proved the highest compatibility of the combination. The decrease in the pollen tube numbers in each part of the pistils, when examined 14 d after pollination, may be caused by digestion of the pollen tube membrane by surrounding pistil tissues.

Open- and self-pollination resulted in only a few pollen tubes reaching the micropyle. Some pollen tubes swelled, bent back, and grew in the opposite direction on the stigma, leading to the slowest and lowest number of pollen tubes penetrating into ovules. From these results it is clear that Zuili stigmas cannot receive the sufficient numbers of potent pollen grains by open-pollination and Zuili plum has incomplete self-incompatibility. To improve the successful cross-pollination by potent pollen grains, interplanting with effective pollinizer plants, such as Black Amber, must be indispensable. Studies of Kaufmane and Rumpunen (2002) on Japanese quince showed that pollen tubes reached the base of the ovary in 2–6 d in a case of a compatible combination, but not until 6–7 d after in an incompatible combination. Yamashita et al. (1990) found that in pears, compatible pollen tubes reached the ovary 3 d after pollination, but incompatible pollen tubes were completely arrested at the stigma.

The importance of normal ovule development for high fruit set in stone fruits has been reported. Alburquerque et al. (2004) reported that apricot cultivars producing good yields had developed mature ovules earlier than those with low productivity, and pollen tubes also grew faster in higher productive cultivars. An anatomical investigation of Zuili ovaries at the petal fall stage revealed that 24.3% of ovules in normal pistils (not double-type pistils) had developed normally. It is generally known that in stone fruit trees, one of the two ovules formed in a pistil usually degenerates before the blooming time (Thompsom and Liu 1973; Pimienta and Polito 1982). The percentage of normally developed ovules counted in this investigation indicated that 29.8% of total ovules degenerated entirely and 45.9% of them were malformed ovules, mainly without embryo sacs in normal-type pistils. The lack of differentiation of the embryo sac has been described as a malformation in apricot (Eaton and Jamont 1964; Lichou et al. 1995) and other species such as avocado (Tomer et al. 1976), litchi (Stern et al. 1996) and peach (Fuss et al. 1990). Such imperfection of ovule development causing poor berry sets has been reported for tetraploid grapes by Okamoto et al. (1984). They investigated the ovule development anatomically and found that 50% to 80% of ovules are abnormal or immature even at the full bloom stage in cv. Kyoho and Pione, usually resulting in a very poor berry set. In our anatomical investigation for Zuili pistils, the notable percentages of imperfection of ovules and embryo sacs are thought to be a cause of the poor fruit setting. On the other hand, poor cultural conditions, as reflected by reduced vigor of apple trees and flowers, or reduced N levels, cause suppression of embryo sac development and reduction of ovule longevity, as well as slower pollen tube growth and reduced periods of stigma receptivity (Williams 1963). Poor fruit set has been attributed to a lack of adequate pollination or wet and cold weather conditions during bloom in apricot (Eaton 1959) and prune (Thompson and Liu 1973). It has been shown that embryo sac degeneration can account for the poor fruit set in sweet cherry (Bradbury 1929; Child 1966).

A particularly interesting finding in this work is the high percentage of double-type pistils in “Zuili”. Unfortunately, in the double pistils, the percentage of normally developed ovules was as low as 8.9%, which indicates the poor fruit set potential of such abnormal pistils. As noted previously, the high rate of double pistils, as 40% of total flowers, must be one of the major reasons for the poor set of Zuili plums in Jiaxin.

Development of double-type pistils in cherry trees has been also reported. Beppu and Kataoka (1999, 2000) and Beppu et al. (2001) noted that the high temperature conditions during a summer season, corresponding to the time of sepal initiation, may cause the abnormality. We already investigated the time of pistil development in Zuili flower buds and determined it to be around early autumn (unpublished data). For preventing the double-pistil development in “Zuili” trees, it might be effective to shade fruiting twigs during summer and early autumn.

This is the first study involving pollen tube growth and compatibility relationships of the Zuili genotype. The results have important implications for plantation establishment for this crop. The mechanism of poor crop in Zuili has been elucidated with two hypotheses. One is the inhibition of pollen tube growth at the base of stigma or in the style, and the other is the developmental arrest of embryo sac or endosperm. As a result of our experiment, pollen germination on stigma and pollen tube growth in the style was inhibited by self-pollination compared with cross-pollination. Thus, we assumed that self-incompatibility occurred before the pollen tube reached the ovary. Many malformations and degenerations of ovules such as those without embryo sacs and double pistil development might cause big problems for Zuili production.

Materials and Methods

(Handling editor: Hong-Wen Huang)