Production of Male Flowers on Rambutan (Nephelium lappaceum L.) Trees in Hawai'i Andrea M. Kawabata, Mike A. Nagao and Laura K. Awong University of Hawaii at Manoa, Beaumont Agricultural Research Center, 875 Komohana St. Hilo, HI 96720 ABSTRACT Production of deformed rambutan fruits has been attributed to poor pollination and an insufficient amount of male flowers in Hawai'i orchards. The objectives of this study were to monitor the development of hermaphroditic functionally male (HFM) flowers on trees of the ‘Silengkeng’ cultivar, determine if naphthaleneacetic acid (NAA) could induce development of HFM flowers on cultivars grown in Hawai'i, identify the stage at which floral buds are responsive to NAA, and document the viability of pollen from HFM flowers. Results showed that with the exception of ‘Silengkeng’, cultivars planted in Hawai'i produced few or no HFM flowers for pollination. ‘Silengkeng’ panicles produced large numbers of HFM flowers throughout the anthesis period which made it a suitable pollinator cultivar. NAA applied to the ‘R7’, ‘R9’, ‘R134’, ‘R162’, ‘R167’, ‘R156 Red’, ‘R156 Yellow’, ‘Binjai’, ‘Jitlee’ and ‘Rongrien’ panicles composed of predominantly hermaphroditic functionally female (HFF) flowers stimulated development of HFM flowers within 6 days after treatment. By 12 days after treatment, production of HFM flowers was decreased. Only floral buds at a stage in development where the apex of the pistil began protruding through the unopened sepals were responsive to NAA. Pollen obtained from naturally produced HFM flowers on ‘Silengkeng’ panicles and from HFM flowers produced after NAA treatment was viable and germinated within 24 hours after incubation in a culture medium. Incorporating ‘Silengkeng’ and male trees in the orchard or treating HFF panicles at the appropriate stage of development can be strategies for increasing male flowers and fruit set and reducing the development of fruit without arils. KEYWORDS: flowering, fruit set, NAA, parthenocarpic fruit. INTRODUCTION Poor fruit set and development of fruits that lack an aril has been a recurring problem in many rambutan orchards in Hawai'i. Studies in affected orchards showed that exclusion of insect pollinators during anthesis resulted in development of a high percentage of these deformed fruits and indicated that insufficient pollination was the cause of the problem (Nagao et al., 2002). Production of deformed fruits was similar to reports from Thailand where parthenocarpic type fruits fail to develop fully and contain little aril (Kosiyachinda and Salma, 1987). During rambutan flowering, 3 types of flowers are produced, and trees are classified into 3 groups based upon flower morphology (Tindall et al., 1994); Valmayor et al., 1970). One group produces only staminate male flowers, which lack a functional ovary and possess 5 to 7 well-developed stamens with anthers that dehisce and shed pollen at anthesis. A second group produces

only hermaphroditic functionally female (HFF) flowers which set fruit but do not shed pollen. At anthesis HFF flowers have a well-developed ovary with a prominent bifid stigma extending beyond stamens that possess anthers which do not dehisce. A third group of trees will produce panicles with both HFF and hermaphroditic functionally male (HFM) flowers. The HFM flowers closely resemble HFF flowers, but have well developed stamens often extending beyond an under-developed pistil. Anthers on these flowers dehisce and shed pollen. A shortage of male flowers results in poor fruit set and development of parthenocarpic type fruits that contain little aril (Kosiyachinda and Salma, 1987; Salakpetch, 2005). Establishing male trees into the orchard, suspending male panicles from branches during flowering and using napthaleneacetic acid (NAA) to increase male flower development have been used to aid pollination in Thailand (Salakpetch, 2005). Application of NAA to panicles with HFF flowers during early anthesis has been shown to induce development of HFM flowers, and is essential for fruit set and production in some Thailand growing locations (Kosiyachina and Salma, 1987; Tindall et al., 1994). Experiments with ‘Jitlee’ and ‘Binjai’ in Hawai'i have also shown that 90 mg/L NAA stimulated production of HFM flowers (Nagao et al., 2002), however, the response of other cultivars planted in Hawai'i and the stage of development at which individual flowers are responsive to NAA have not been determined. Also the viability of pollen from NAA induced HFF flowers has not been documented. Most cultivars produce a high percentage of HFF flowers and a low percentage of HFM flowers which may amount to only 0.05 to 0.9% of the flowers found on a panicle (Nakasone and Paull, 1998; Tindall et al., 1994). Some orchards in Hawai'i have established the ‘Silengkeng’ cultivar to aid in pollination, but its ability to produce HFM flowers under Hawai'i growing conditions has not been documented. The objectives of this study were to monitor development of HFM flowers on ‘Silengkeng’ trees, to determine if NAA could induce development of HFM flowers on cultivars grown in Hawai'i, to identify the stage at which floral buds are responsive to NAA treatments and to document the viability of pollen produced on HFM flowers. MATERIALS AND METHODS HFM flower development on ‘Silengkeng’ panicles. To observe occurrence of HFM flowers, 22 panicles distributed among four ‘Silengkeng’ trees were randomly selected in an orchard in Onomea, HI (115 m elevation) in 2003 July. Trees were at least 12 years-old, and each panicle was monitored from the onset to the termination of anthesis by observing the number of newly opened HFM flowers at 3 to 4 day intervals. Response of cultivars to NAA. Individual panicles were sprayed to runoff with a hand-held sprayer containing distilled water or an aqueous solution of 90 mg/L potassium salt of NAA (K+NAA). Treatments were applied when approximately 10% of the flowers on the panicle were at anthesis based on a visual assessment of each panicle. The experiment was conducted between 2004 June to August in orchards at Onomea, Kurtistown, (250 m elevation) and Kea´au, HI (75 m elevation). A minimum of 15 replicate panicles distributed on at least 3 trees of each cultivar were treated. Because flowering among rambutan trees in Hawai'i is non-synchronous (Kawabata et al., 2007), for an individual cultivar an equal number of treated and control panicles was sprayed on

each tree, but the number of replicate panicles/tree was variable. Table 1 shows the cultivars, location of the experiments, number of replicate panicles treated, number of replicate panicles/tree, and number of trees treated. The number of newly-opened HFM flowers was recorded at 0, 4, 6, 8, 10, and 12 days after treatment. Stage of flower development responsive to NAA. In 2004 August, 18 replicate panicles equally distributed on 3 ‘Jitlee’ trees in Onomea, HI were sprayed with 0 or 90 mg/L K+NAA to run-off with a hand-sprayer. Individual flowers on each panicle were monitored by marking a section of the panicle with indelible ink and photographing the flowers at 0, 3 and 6 days after treatment to identify the stage of flower development responsive to NAA treatment. Pollen viability of ‘Silengkeng’ and NAA induced HFM flowers. ‘Binjai’, ‘R156 Red’, ‘Jitlee’, ‘Rongrien’, ‘R7’, ‘R9’, ‘R134’, ‘R167’, ‘R162’, and ‘R156 Yellow’ panicles were treated with 90 ml/L K+NAA between 2004 August and September. At 7 days after treatment, panicles were removed from the tree and transported to the laboratory in enclosed polyethylene bags containing moistened paper towels. Pollen from 5 HFM flowers produced on untreated ‘Silengkeng’ panicles and from treated panicles of each cultivar was tested for germination. Flowers with dehisced anthers were removed from each panicle. A drop of germination medium was placed on a microscope slide and pollen grains were removed from 2 anthers per flower and placed in the drop of germination medium. The medium consisted of a modified Brewbaker and Kwack (1963) solution containing 50 ppm H3BO3, 150 ppm Ca(NO3)2.4H2O, 100 ppm MgSO4.7H2O, 50 ppm KNO3, and 5% sucrose. The bottom of 100 x 10 mm glass Petri dishes was fitted with filter paper moistened with distilled water. The slide was placed on the moistened filter paper, the dish covered, and incubated for 24 to 48 hours at 22.8°C. Pollen was considered viable, if pollen germination occurred and pollen tubes were visible within 48 hours. RESULTS AND DISCUSSION Anthesis on individual ‘Silengkeng’ panicles occurred over a 17 to 161 day period, and the mean number of days for duration of anthesis for panicles was 81 days. Figure 1 shows that production of HFM flowers occurred at highest frequency during the initial third of the anthesis period, with smaller numbers produced during the remaining anthesis period. At the peak of HFM flower production, approximately 84 HFM flowers were observed on each panicle interspersed with HFF flowers. The duration of flowering for ‘Silengkeng’ panicles was much longer than observed on other cultivars planted in Hawai'i (Kawabata et al., 2007) and in Thailand (Tongumpai et al., 1980), and production of HFM flowers on ‘Silengkeng’ panicles was more numerous than reported for other cultivars. Studies in the Philippines and Malaysia (Tindall et al., 1994) showed that the ‘Maharlika’ and ‘Seejonja’ cultivars also produced both HFM and HFF on the same panicle. Approximately 99.94% of the flowers on ‘Maharlika’ panicles were functionally female, while the remaining flowers were functionally male. The percentage of HFF flowers on ‘Seejonja’ panicles was 99.55% at bloom. Tindall et al. (1994) also reported that generally between 200 to 800 HFF flowers are borne on each panicle and that HFM flowers make up about 0.05 to 0.9% of the flowers. The high frequency and the

continued production of HFM flowers on ‘Silengkeng’ panicles over the entire anthesis period confirm that ‘Silengkeng’ may serve as a suitable source of pollen for fruit set. All cultivars treated with 90 mg/L NAA produced HFM flowers by 4 to 6 days after treatment (Table 2). HFM flower production was greatest between 6 and 8 days after treatment and progressively decreased at 10 and 12 days. Since the appearance of HFM flowers decreased at 12 days after NAA treatment, re-application to adjacent panicles will be necessary to sustain HFM flower production throughout the anthesis period which can extend over several weeks (Kawabata et al., 2007). Within each location some cultivars tended to have a greater response to NAA. At Onomea, ‘Rongrien’ produced the greatest number HFM flowers (124.7 flowers per panicle) while ‘R156 Red’ produced the fewest HFM flowers. Elevation also may have had an effect since the ‘R9’ cultivar at Kurtistown was not as responsive as at Onomea. Since male trees do not produce fruits, some growers in Thailand tend to remove these trees, and the result is an insufficient amount of pollen available to fertilize the HFF flowers. NAA concentrations between 40 and 160 mg/L are then used to insure fruit set and are essential for production in some areas (Kosiyachina and Salma, 1987; Lam and Tongumpai, 1987). Sprays are applied to panicles that are 1.0 meter apart and when onethird of the flowers on the panicle are open (Tindall et al., 1994). HFM flowers are visible 5-7 days later. After the pollen is shed from NAA treated panicles, panicles are pruned off and newly emerging panicles will produce normal HFF flowers (Salakpetch, 2005). HFM flowers were not produced on the control panicles except for ‘R156 Yellow’ and ‘R134’ at Onomea, and in both cultivars natural production of HFM flowers was very low (Table 2). The small number of HFM flowers produced by these cultivars without NAA is probably too few to adequately pollinate the large number of HFF flowers. Figure 2 shows ‘Jitlee’ flower buds on the day of treatment and at 3 and 6 days after NAA application. Buds on which the apex of the pistil began protruding through the unopened sepals responded to NAA and developed into HFM flowers that were at anthesis by 6 days after treatment. HFF flowers that were at partial or full anthesis and buds that were tightly closed at the time of treatment did not respond to NAA. These buds later developed into HFF flowers or abscised from the panicles. Untreated HFF flowers at anthesis possess a pistil topped with a bifid stigma which is surrounded by 6 short stamens arising from the base of the bilocular ovary. Stamens do not extend beyond the stigma, and anthers do not dehisce and brown by the second day after anthesis. In HFM flowers, NAA stimulated elongation of the stamens and promoted anther dehiscence while suppressing pistil development. NAA application to individual panicles should be timed to coincide with a period when there is an abundance of floral buds that are at a stage responsive to NAA. At 10% anthesis, panicles possess numerous floral buds at this stage of development. Germination was observed with pollen obtained from naturally occurring HFM flowers on ‘Silengkeng’ panicles and from HFM flowers produced after NAA treatment for all cultivars tested. Germination of pollen grains and growth of pollen tubes could be seen 16 to 48 hours after pollen was incubated in the germination medium (Figure 3). There appeared to be a population effect during pollen germination, because germination percentage tended to be lower when fewer pollen grains were incubated in the medium.

Germination tests confirmed that viable pollen was produced by naturally occurring HFM flowers from ‘Silengkeng’ panicles and from NAA induced flowers. Our attempts to germinate pollen taken from anthers on HFF flowers were unsuccessful and support the conclusion that HFF flowers only function as female flowers (Tindall et al., 1994). Bagging experiments in Malaysia and Hawai'i to exclude pollinators reinforce the importance of pollination for rambutan production since poor pollination will result in reduced fruit set and production of deformed fruit (Lim, 1984; Nagao et al., 2002). With the exception of ‘Silengkeng’, most cultivars in Hawai'i produce very few or no HFM flowers to adequately supply pollen in a large orchard. Treating panicles with NAA will increase production of viable pollen when applied to flower buds at the appropriate stage of development. Based on our observations, smaller, more compact panicles seemed to be less responsive to NAA treatment compared to large, more elongated panicles since some smaller panicles did not produce HFM flowers after NAA treatment. Rambutan flowering is initiated following a 2-4 week period of drought stress, and flowering intensity is thought to be closely related to intensity and duration of the water stress (Nakasone and Paull, 1998; Whitehead, 1959). Our observations indicate that during the cooler flowering season (April to May), smaller compact panicles are more common than during the warmer flowering season (July to August) in Hawai'i. Therefore selection of suitable panicles for treatment as well as the timing of NAA application will be important for an effective NAA response. ACKNOWLEDGEMENTS We gratefully acknowledge the support of Onomea Orchards and Plant-It Hawaii for the use of their orchards. This research was supported with grants from the Hawaii Department of Agriculture and the Hawaii Farm Bureau Federation.

LITERATURE CITED Brewbaker, J.L. and B.H. Kwack. 1963. The essential role of calcium in pollen germination and pollen tube growth. Am. J. Bot. 50(9):859-865. Kawabata, A.M., M.A. Nagao, T. Tsumura, D.F. Aoki, K.Y. Hara and L.K. Pena. 2007. Phenology and fruit development of rambutan (Nephelium lappaceum L.) grown in Hawai'i. J. Hawaiian Pacific. Agric. 14:31-39. Kosiyachinda, S. and I. Salma. 1987. Changes in rambutan during growth and development. In: Rambutan: Fruit Development, Postharvest Physiology, and Marketing in ASEAN. Kuala Lumpur, Malaysia, 16-27. Lam, P.F. and P. Tongumpai. 1987. Preharvest factors affecting postharvest quality of rambutan. In: Rambutan: Fruit Development, Postharvest Physiology, and Marketing in ASEAN. Kuala Lumpur, Malaysia, 27-32. Lim, A.L. 1984. The reproductive biology of rambutan, Nephelium lappaceum L. (Sapindaceae). Gard. Bull. Sing. 37(2):181-192. Nagao, M.A., H.M. Leite, A.M. Kawabata, and A.Y. Terada. 2002. Abstract: Update of tropical fruit research in Hawai´i. In: Proceedings of the 12th Annual International Tropical Fruit Conference. Keauhou Kona, Hawaii, 8. Nakasone, H.Y. and R.E Paull. 1998. Tropical fruits. CAB International. Wallingford, UK. Salakpetch, S. 2005. Rambutan production in Thailand. Acta Horticulturae 665:67-72. Tindall, H.D., U.G. Menini and A.J. Hodder. 1994. Rambutan cultivation. Food and Agriculture Organization of the United Nations. Rome, Italy. Tongumpai, P., R. Sethpakdee, and B. Silayoi. 1980. Effect of some growth regulators on sex expression of ‘Seechompoo’ rambutan. J. of Hort. 15:31-38. Valmayor, R.V., C.O. Palencia, H.B. Aycardo, and D.B. Mendoza. 1970. Growth and flowering habits, floral biology and yield of rambutan (Nephelium lappaceum Linn.). Philippine Agriculturalist 54(7/8):359-374. Whitehead, C. 1959. The rambutan, a description of the characteristics and potential of the more important varieties. Malayan Agr. J. 42:53-75.

Table 1. Location and cultivars treated with K+NAA. Cultivar

Location

Replicate Replicate panicles panicles/tree

R167 R9 Binjai Jitlee R162 R134 R9 R156 Red Rongrien R7 R156 Yellow

Kurtistown Kurtistown Keaau Keaau Keaau Onomea Onomea Onomea Onomea Onomea Onomea

15 15 15 17 22 16 15 18 17 18 16

No. trees treated

5 5 5 4-8 5-10 2-6 2-5 3-5 5-7 2-8 2-8

3 3 3 3 3 5 4 5 3 4 3

Table 2. Production of HFM flowers on panicles treated with 0 (control) and 90 ml/L K+NAA at 0, 4, 6, 8, 10, and 12 days after treatment. No. HFM flowers/panicle

a

Cultivar

Location

R167

Kurtistown

R9

Kurtistown

Binjai

Keaau

0

4

6

8

10

12

NAA

NAA

NAA

NAA

NAA

NAA

0

0

16.5 ± 5.5a

28.9 ± 9.0

17.6 ± 5.8

7.8 ± 2.7

0

0

1.2 ± 0.4

13.7 ± 2.3

5.1 ± 2.1

0

0

23.3 ± 10.6

28.5 ± 8.0

19.4 ± 3.7

6.4 ± 1.9

1.3 ± 0.4 15.2 ± 3.1

Jitlee

Keaau

0

10.5 ± 2.8

65.8 ± 17.1

84.8 ± 13.1

65.0 ± 9.0

R162

Keaau

0

2.7 ± 1.9

50.8 ± 19.6

40.5 ± 14.5

22.5 ± 9.8

12 ± 5.0

R134

Onomea

0.1 ± 0.1

2.7 ± 1.5

37.0 ± 16.0

36.5 ± 14.4

18.3 ± 9.0

5.6 ± 4.3 9.2 ± 2.1

R9

Onomea

0

0

50.6 ± 5.3

51.3 ± 6.2

20.9 ± 4.1

R156 Red

Onomea

0

0

4.5 ± 1.7

9.6 ± 2.7

6.7 ± 1.8

4.4 ± 1.6

Rongrien

Onomea

0

18.5 ± 9.5

106.2 ± 32.8

111.2 ± 24.9

88.9 ± 19.9

41.6 ± 13.0

R7

Onomea

0

0.6 ± 0.4

27.4 ± 7.3

81.7 ± 18.2

47.3 ± 12.0

17.0 ± 6.3

R156 Yellow

Onomea

0.8 ± 0.3

13.6 ± 5.1

37.3 ± 7.8

67.9 ± 13.5

52.1 ± 11.5

19.8 ± 6.6

Control

Control

Control

Control

Control

Control

R134

Onomea

0.1 ± 0.1

0.3 ± 0.3

0.4 ± 0.3

0.3± 0.2

0.1± 0.1

0

R156 Yellow

Onomea

0.2 ± 0.1

0.4 ± 0.3

0.1 ± 0.1

0

0

0

Each value represents the mean ± SE

Figure 1. Frequency of naturally produced HFM flowers on ‘Silengkeng’ panicles observed at 3 to 4 days intervals from the onset to the termination of anthesis. Error bars represent ± SE. n=22.

80

60

40

20

0 0 3 7 10 14 17 21 24 28 31 35 38 42 45 49 52 56 59 63 66 70 73 77 80 84 87 91 94 98 101 105 108 112 115 119 122 126 129 133 136 140 143 147 150 154 157 161

No. of HFM Flowers per Panicle

Figure 1. Frequency of naturally produced HFM flowers on ‘Silengkeng’ panicles 120 observed at 3 to 4 days intervals from the onset to the termination of anthesis. Error bars represent ± SE. 100

Days After Anthesis

Figure 2. Development of ‘Jitlee’ rambutan flower buds at 0, 3 and 6 days after treatment with 90 mg/L NAA. Arrows show progressive development of buds responsive to NAA.

0 day

3 days

O day

6 days

3 days

6 days

Figure 3. Germination of pollen from HFM flowers from untreated ‘Silengkeng’ panicles (a) and from HFM flowers developing after 90 mg/L K+NAA treatment of ‘Rongrien’ panicles (b).

a

b