ISSN 0216-0919 Jakarta
2009 Pages
85 -165
I
J. For. Res Vol. 6 No. 2Ministry of Forestry
Forestry Research and Development Agency
Jakarta Indonesia
Accredited B by the Indonesian Institute of Sciences No. 683/D/2008 (115/ Akred-LIPI/P2MBI/06/2008) All inquiries and manuscripts should be sent to the :
Journal of Forestry Research Telephone : (0251)-7522638 Sub Bagian Data clan Informasi Fax : (0251)-7522638 Sekretariat Badan Litbang Kehutanan E-mail : [email protected] JI. Gunung Batu No. 5 Bogor 16610 Website : http://www.forda-mof.org
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©2009, Forestry Research and Development Agency, Indonesia
Journal of Forestry Research is previously published as Forestry Research Journal (2000 - 2001). This Journal is published in one volume of two issues per year by the Forestry Research and Development Agency, Indonesia.
The journal publishes primary research findings and synthesized articles containing significant contribution to science and its theoretical application in forestry in Indonesia. Overseas works relevant to Indonesian conditions may be accepted for consideration.
Editorial Board
Dr. .Irnayuli R. Sitepu (Forest Microbiology) Dr. LB. Putera Parthama (Forest Biometrics) Dr. Taulana Sukandi (Agroforestry, Community Forestry)
Prof. (Ris) Dr. Wayan Laba (Plant Pests and Diseases) Dr. Sofyan P. Warsito (Forest Resource Economics) Dr. Han Roliadi (Forest Products Technology and Chemistry)
Dr. Sri Nugroho Marsoem (Wood Science) Prof. Dr. Ani Mardiastuti (Conservation and Biodiversity)
Dr. Iskandar Zulkarnaen Siregar (Forest Genetics) Dr. Anto Rimbawanto (Molecular Biology)
Dr. Chay Asdak (Forest Hydrology) Dr. Kade Sidiyasa (Taxonomy)
Managing Editors
Head of Research Cooperation and Information Division Head of Data and Information Sub Division
Dr. Haruni Krisnawati Ratih Damayanti
Dian Anggraini Raditya Arief
Bintoro
Journal of For estry Rese a rch
Vol. 6 No. 2, 2009
Prof Dr. Yusuf Sudo Hadi Forest Product Technology
Bogor Agricultural University, Indonesia Dr. Sri Wilarso Budi
Biotechnology
Bogor Agricultural University, Indonesia Dr. Kuswata Kartawinata
Botany and Forest Ecology
United Nations Educational, Scientific and Cultural Organization (UNESCO) Dr. Irsyal Yasman
Silviculture, Ecophysiology PT. Inhutani I, Indonesia Prof Dr. Endang Suhendang
Forest Management, Forest Biometrics Bogor Agricultural University, Indonesia Dr. Edi Soenarjo
Entomology
Indonesian Center for Food Crops Research and Development, Indonesia Dr. Aulia Aruan
Forest Planning, Tropical Plantation Management PT. Riau Andalan Pulp and Paper, Indonesia Anonymous
Tree Breeding
Kyushu University, Japan Reviewers
- 1-..,
l
DOMINANT HEIGHT AND SITE INDEX MODELS FOR Acacia mangium Willd. PLANT A TIO NS
Haruni Krisnawati, Yue Wang, Peter K. Ades, and Ian W. Wild... 148 THE EFFECT OF ARBUSCULAR MYCORRHIZAL FUNGI AND SLOW RELEASE FERTILIZER ON THE GROWTH OF Alstonia scholaris
(L.)
Br. SEEDLINGS IN THE NURSERYRagil S.B. Irianto .. . .. .. . 139 POLLEN TUBES GROWTH FOLLOWING SELF- AND CROSS- POLLINA TION IN Melaleuca alternifolia (Maiden & Betche) Cheel Liliana Baskorowati . .. . . .. .. . . 126 PARASITES AND PREDATORS OF Laccifer lacca Kerr ON LAC CULTURE IN EAST SUMBA, EAST NUSA TENGGARA
Sujarwo Sujatmoko .. . .. . .. .. 119 GROWTH-PROMOTING PROPER TIES OF BACTERIA ISOLATED FROM DIPTEROCARP PLANTS OF ACIDIC LOWLAND TROPICAL PEAT FOREST IN CENTRAL KALIMANTAN, INDONESIA
Irnayuli R. Sitepu, Y asuyuki Hashidoko, Erdy Santoso, and Satoshi
Tahara 96
AN OPTIMUM DESIGN FOR SEEDLING SEED ORCHARDS TO MAXIMIZE GENETIC GAIN: AN INVESTIGATION ON SEEDLING SEED ORCHARDS OF Eucalyptus pellita F. Muell
Budi Leksono, Susumu Kurinobu, and Yuji Ide... 85
Contents
Titles Pages
Journal of Forestry Research
Vol. 6 No. 2, 2009
ISSN 0216-0919
115/ Akred-LIPI/P2MBI/06/2008
GROWTH-PROMOTING PROPERTIES OF BACTERIA ISOLA TED FROM DIPTEROCARP PLANTS OF ACIDIC LOWLAND TROPICAL PEAT FOREST IN CENTRAL KALIMANTAN, INDONESIA
(SIFAT PEMACU PERTUMBUHAN DARI BAKTERI YANG DJISOLASI DARI JENIS DIPTEROKARPA DI HUTAN GAMBUT TROPIS DATARAN RENDAH DI KALIMANTAN TENGAH, INDONESIA)
Irnayuli R. Sitepu, Yasuyuki Hashidoko, Erdy Santoso, dan Satoshi Tahara
Penelitian ini bertujuan untuk mcnyeleksi bakreri yang memiliki sifat-sifat sebagai pemacu pertumbuhan. Bakreri diisolasi dari tanah (rhizos6r) dan akar (rhizoplan) jenis-jenis dipterokarpa, yaitu Sborea teysmanniana, S. paruiflora, S.
balangeran, S. stenoptera, Dipterocarpus sp., dan Hopea sp. yang tumbub di hutan rawa gambut dataran rendab di Kalimantan Tengah. Sebanyak 71 bakteri yang dirurnbuhkan pada medium gel lunak Winogradsl..-y tanpa nitrogen, discleksi secara in vitro.
Seleksi meliputi kemampuan dalam melarutkan P, menambat N,, dan/arau memfosilitasi ekrornikoriza. Hampir seluruh bakreri menunjukkan kemampuan sebagai pemacu perturnbuhan, namun 14 diantaranya memiliki e6siensi terbaik. Pelarut fosfat paling efisien adalah Erunnia spp. CK23, CK24 dan CKIO, Roseate/es sp.
CK15, Rbizobium sp. CK19, Burkbolderia sp. CK52, NI CK36, NI CK42, NI CK53, dan NI CK54. Penambat N, tertinggi adalah Azospirillum sp, CK26, Burkbolderia sp. CK32, dan NI CK4. Sedangkan Cbromobacterium sp. CK8 adalah bakteri yang potensial sebagai pemacu pertumbuhan fungi ektornikoriza, Laccaria sp. Studi ini meounjukkan bahwa Diprerocarpaceae merupakan habitat bagi beragam jenis genus bakteri dan kandidat pemacu pertumbuhan yang akan diuji lanjut unruk bioreforestasi lahan terdegradasi.
Kata kunci: Pelarut P, penambat N,, ektomikoriza, Erwinia spp., Azospiri/111m sp., Chromobacteri11m sp., biorcforestasi
UDC/ODC 630"151.42(594.73)
PARASITES AND PREDA TORS OF Laccifer lacca Kerr ON LAC CULTURE IN EAST SUMBA, EAST NUSA TENGGARA
(PARA SIT DAN PREDATOR DARI Laccifer lacca Kerr PADA BUDIDA YA LAC DI SUMBA TIMUR, NUSA TENGGARA TIMUR)
Sujarwo Sujatmoko
Penelitian ini berrujuan unruk mengindenti6kasi parasit dan predator yang menyerang lac di Sumba Timur, Nusa Tenggara Timur. Serangga lac dikumpulkan dari Hambapraing, Mbatakapidu dan Kuta, serta diidenti6kasi di Laboratorum Entomologi Dasar, Universitas Gadjah Mada, Yogyakarta. Hasil idenrifikasi menunjukkan bahwa predator baru lac adalah Dolichoderus tboracicus Smith (Hymenoptera: Formicidae) dan Catoblemma sumbavensis Hampson (Lepidoptera: Noctuidae). Parasit lac yong diternukan adalah Coccophagus tscbircbii Madhihassen (Hymenoptera: Aphelinidae) dan Enpelmus tacbardiae Howard (Hymenoptera: Euphelmidae). Tingginya populasi predator dan parasir diduga akibat minimnya perawatan dan sanitasi di tempat tumbuh lac. Studi lanjur dibutuhkan udtuk mengetahui mekanisme biperparasit di lokasi budidaya lac.
Kata kunci: Laccifer lacca Kerr, parasit dan predator lac., Sumba Timur UDC/ODC 630"181.525 UDC/ODC 630"181.525
Keywords given are free terms. Abstracts may be reproduced without permission or charge Vol. 6 No. 2, 2009 ABSTRACTS
ISSN 0216-0919
AN OPTIMUM DESIGN FOR SEEDLING
SEED ORCHARDS TO MAXIMIZE
GENETIC GAIN: AN INVESTIGATION ON SEEDLING SEED ORCHARDS OF
£11calyp111s pellita F. Muell
(DESAIN YANG OPTIMUM UNTUK
KEBUN BENIH SEMAI DALAM RANGKA PENINGKA TAN PERO LEHAN GENETIK:
PENELITIAN P ADA KEBUN BENIH SEMAI
£11calyp1m pellita F. Muell)
Budi Leksono, Susumu Kurinobu, dan Yuji Ide Desain yang optimum untuk pembangunan kebun benih semai (KBS) dalam rangka peningkatan gcnetik dilakukan untuk mengevaluasi desain yang digunakan pada gencrasi penama. Analisis dilakukan pada KBS Eucalyptns pellita di Kalirnanran Selaran dan Sumatera Selaran terhadap tinggi tanaman umur 1 · 5 tahun. Estimasi dilakukan terhadap tiga komponen varian (varian famili, varian galat plot dan varian di dalam plot) pada tiga level heritabilitas (rendah, sedang dan tinggi).
Jumlah famili yang optimum pada heritabiliras sedang sebesar 40-50 dengan jumlah pohon per plot anrara 6-8 untuk KBS scluas 2 ha dengan 10 blok. Hasil ini menunjukkan bahwa desain KBS E. pellita mendekati desain optimum unruk pembangunan KBS dengan dua tabapan seleksi (seleksi di dalam plot dan seleksi antar famili).
Kata kunci: Eucalyptus pellit a, perolehan generik, desain optimum, ukuran plot, kebun benih semai
J our n al of Fores t ry R esear ch
f
UDC/ODC 630''232.322
THE EFFECT OF ARBUSCULAR
MYCORRHIZAL FUNGI AND
SLOW RELEASE FERTILIZER ON THE GROWTH OF Alstonia scbolaris (L.) Br. SEEDLINGS IN THE NURSERY (PENGARUH FUNGI MIKORIZA
ARBUSKULA DAN PUPUK
LEPAS LAMBAT TERHADAP
PER TUMBUHAN BIBIT Alston in scbolarts (L.) Br. DI PERSEMAIAN)
Ragil S.B. Irianto
Tujuan penelician adalah untuk mengerahui pengaruh fungi mikoriza arbuskula (FMA) dan pupuk lepas larnbat unruk memacu pertumbuhan bibic Alstonia scbolaris (L.) Br. di persemaian. Rancangan acak kelompok lengkap clengan dua fakcor, yairu jenis FMA clan dosis pemupukan yang masing-masing cliulang ernpat kali. Hasil penelitian menunjukkan bahwa kombinasi inokulasi FMA ( Glomus aggregatum ) clan pemupukan (0,4 g) dapat meningkarkan cinggi, diameter, dan berar kering bibit, yaitu berturut-turur 95-521%, 43-237%, dan 571-2.735% dibandingkan dengan bibic yang cidak diinokulasi. Penelician ini perlu dirindaklanjuti dengan penelician pada skala lapangan untuk menunjang Gerban.
Kara kunci: Glomus aggregawm, inokulasi, berat kering, rehabilicasi lahan UDC/ODC 630*181.521
POLLEN TUBES GROWTH FOLLOWING SELF-AND CROSS- POLLINA TION IN Melaleucn alternifolia {Maiden & Berche) Chee!
{PERTUMBUHAN TABUNG SERBUK SARI SETELAH
PENYERBUKAN SENDIRI DAN PENYERBUKAN SILANG PADA Melalenc« altemifolia (Maiden & Betche) Cheel)
Liliana Baskorowari
Penelirian ini bertujuan unruk meogetahui penumbuhan rabung serbuk sari setelah penyerbukan sendiri clan penyerbukan silang dari beberapa individu pobon Melaleuca alternifolia (Maiden
& Betche) Chee! di dua kebun benih M. alternifolia Wyalong Barat,
New South Wales, Australia pada tahun 2004 clan 2005. Jumlah tabung serbuk sari diamati menggunakan mikroskop floresen pada hari ke-4, -7, -9 clan -14 serelah penyerbukan. Penetrasi bakal biji oleh tabung serbuk sari diamati serelah 14 bari penyerbukan.
Hasil penelitian menunjukkan bahwa jumlah tabung serbuk sari pada tangkai purik sangac bervariasi antara tabung serbuk sari dari penyerbukan sendiri clan penyerbukan silang. Jumlah rabung serbuk sari pada tangkai putik penyerbukan sendiri lebih sedikit daripada penyerbukan silang, dan jumlahnya menurun drasris saat interval pemanenan diringkackan, yaitu mulai 4 hari setelah penyerbukan.
Mekanisme keridakcocokan kawin sendiri dapar rerjadi pacla M.
alternifolia . Turunnya jumlab rabung serbuk sari pada bagian bawah cangkai purik menunjukkan bahwa kecidakcocokan kawin sendiri terjadi pada ujung purik maupun tangkai pucik. Penolakan ca bung serbuk sari basil penyerbukan sendiri pada level ovarian dan konsekuensi rendahnya cingkatan acau incoleransi dari penyerbukan sendiri menghasilkan cingginya cingkac penyerbukan silang pacla M.
alternifolia.
Kaea kunci: Me/11/e11c11 alternifolia, ca bung serbuk sari, keridakcocokan kawin sendiri, penyerbukan sendiri, penyerbukan silang UDC/ODC 630*541
DOMINANT HEIGHT AND SITE INDEX MODELS FOR Acacia mangium \'ililld. PLANTATIONS {MODEL PENDUGA PENINGGI DAN INDEKS TEMP AT TUMBUH HUT ANT ANAMAN Acacia manginm
\'ilild.)
Haruni Krisnawari, Yue Wang, Peter K. Ades, dan Ian W. Wild
Penelirian ini bercujuan uncuk mendapatkan model penduga peninggi clan indeks ternpat cumbuh yang kompacibel uncuk hucan tanaman Acacia mangium \'ililld. Tujuh persamaan aljabar diferensial (ADE) yang dicurunkan dari model-model Chapman-Richards, Lundqvist-Korf dan McDill-Amateis telah dianalisis clan dievaluasi secara numerik dan grafis, dengan pertimbangan starisrik dan biologis. Hasil analisis menunjukkan bahwa model Lundqvisr-fungsi Korf yang memiliki parameter asymptote mernberikan basil dugaan peninggi yang paling akurat dan kurva polimorfik indeks rernpar tumbuh yang dihasilkan paling memadai dibandingkan dcngan enam model lainnya. Umur indeks yang paling akurat untuk aplikasi adalah 6 rahun. Model yang dihasilkan lebih fleksibel dalam menduga peninggi dan indeks rempar rumbuh dibandingkan dengan model-model sebelumnya unruk huran tanaman A. mangium.
Kata kunci: Kualiras rempar tumbuh, peninggi, persamaan aljabar diferensial, polimorfik, Acacia mangium
mer (th( dur labc
A.
II.
IS C
sell Mc
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126
I;
1 Center for Forest Biotechnology and Tree Improvement Research, JI. Palagan Tentara Pelajar Km. 15, Purwobinangun, Pakem, Sleman, Y ogyakarta, Indonesia. E-mail: [email protected].
Self-incompatibility is a common characteristic of many flowering plants (Bawa, 1974; Bawa and Beach, 1983). Self-incompatibility may result from a variety of mechanisms, such as the failure of self pollen grains to adhere to the stigma or the failure of pollen tubes to grow down the style (de Nettacourt, 1977; Richard, 1997). There have been reported of more than one self-incompatibility mechanism operating in a species, which may act both the pre- and post-zygotic levels (Sedgley and Griffin, 1989). For example, Eucalyptus spathulata and E. platypus have both pre- and post-zygotic barriers to self pollinated seed production as pollen tube penetration of ovules and ovules fertilization was reduced following self-compared with cross-pollination, and few self-fertilised ovules developed past zygote division (Sedgley and Granger, 1996). The genetic control of a pre-zygotic incompatibility system may be either gametophytic when the incompatibility of phenotype of the pollen is determined by its haploid genome - or sporophytic when the pollen I. INTRODUCTION
Keywords: Melaleuca alternifolia, pollen tubes, self-incompatibility, self-pollination, cross- pollination
Pollen tubes growth following controlled self- and cross-pollination of several individual of trees of Melaleuca alternifolia was observed under fluorescence microscope. Two experiments during 2004 and 2005 flowering seasons were carried out at M. alternifolia seed orchard at West Wyalong, New South Wales (Australia). Pollen tubes number was investigated at 4, 7, 9 and 14 days after pollination by fluorescence microscope. Ovule penetration by pollen tubes was investigated 14 days after pollination. They varied greatly between self- and cross-pollen tubes in the style, with less pollen tube found in the self-pollinated styles than cross-pollinated styles. The mean number of pollen tubes declined significantly as interval to harvesting of the style increased, from a maximum at the 4 days after pollination. The availability of self-pollen tubes grow in the style of M. alternifolia shows that mechanism of self-incompatibility occurs in this species. The reduction of number of pollen tubes in the lower parts of style shows that the self-incompatibility systems operate in the stigma or the style. The rejection of self-pollen tubes at the ovarian level and the consequent low levels or intolerance of selfing maintains a high level of outcrossing in M. alternifolia ..
ABSTRACT Liliana Baskorowati 1
POLLEN TUBES GROWTH FOLLOWING SELF- AND CROSS- POLLINATION IN Melaleuca alternifolia (Maiden & Betche) Cheel
127 Fieldwork to provide the materials of which to be investigated self- incompatibility on the basis of pollen tube growth was undertaken in West Wyalong (the second-generation Seedling Seed Orchard), New South Wales, Australia during the flowering season of November 2005 and November 2006. Subsequent , laboratory work was carried out in the electron microscopic unit (EMU) of the
A. Location
II. MATERIALS AND METHODS
exhibits the incompatibility phenotype of its diploid parent (de Nettacourt, 1977;
Sedgley and Griffin, 1989; Richard, 1997; Franklin-Tong and Franklin, 2003).
Barlow and Forrester (1984), in a study of several compatible intraspecific crosses of Melaleuca species, reported that gametophytic self-incompatibility occurs in these species. They also noted that self-pollen tubes of Melaleuca hypericifolia and M. thymifolia arrest in the ovary, whereas those of M. capitata arrest in the micropyle. Seavy and Bawa (1986) and Sage et al. (1994) characterized the failure of selfed flowers to produce fruits, despite the self-pollen tube growing successfully in the style to the ovary, as the phenomenon of late-acting self-incompatibility.
This has been reported to occur in several Myrtaceous species such as in Eucalyptus regnans (Sedgley et al., 1989), E. cladocalyx and E. leptophylla (Ellis and Sedgley, 1992), E.,nitens (Pound et al., 2003), and E. globulus (Pound et al., 2002a; 2002b).
A recent study onM cajuputi ssp. cajuputi in a seedling seed orchard in Yogjakarta, Indonesia, found that this species has a low level of self-incompatibility (0.035), although some individual trees were completely self-compatible (Kartikawati, 2005).
However, several studies have noted that the mechanisms controlling preferential outcrossing, which reduce inbreeding and promote heterozygosity, vary between species (Sedgley and Griffin, 1989; Potts and Gore, 2000). Results from previous studies of Melaeuca species (Barlow and Forrester, 1984; Kartikawati, 2005) lead
to the hypothesis that self-incompatibility mechanisms operate in M alternifolia.
Melaleuca alternifolia, which flowers annually during spring (Baskorowati, 2008), is considered to have a breeding system that is preferentially outcrossing, although selfing is not uncommon (Butcher et al., 1992; Rosseto et al., 1999; Doran and Moran, 2002). Information on the level and the mechanisms of self-incompatibility is essential to determine the best breeding strategy for M alternifolia. However, published studies on the mating system of M. alternifolia (Butcher et al. 1992;
Rosseto et al. 1999; Doran and Moran 2002) provided very limited information on the level of selfing. The mechanism by which self-incompatibility acts in M.
alternifolia is unknown, as is the impact of selfing on capsule, seed and seedling development. Therefore, this paper reports investigation of pollen tubes growth in M alternifolia pistils following controlled self- and cross-pollination to find out whether self-incompatibility mechanism operates in this species.
Pollen Tubes Growth ... L. Baskorowati
ara Pelajar Km. 15,
oo.com.
wering Rlants from a v~riety
the stigma or 1977; Richard, ity mechanism levels (Sedgley pus have both as pollen tube self-compared ygote division
compatibility phenotype of hen the pollen
llination, cross-
~veral individual I'wo experiments
I
seed orchard atinvestigated at penetration by etween self- and ated styles than antly as interval ollination. The t mechanism of len tubes in the he stigma or the
ent low levels or
6
CROSS-f
che) Ch eelA. First Ex1 The ma>
bottom) of s after pollinati at 1, 2, and 3 III. RESUL'.
Yij = observ µ = overal pl = replica
-CJ = treatm clJ = residu:
Resi for analyzinj (ANOVA) G tubes were tr The number observation, performed w
Then
D. Statistic The sty
(6 : 3 : 1 ab
hours, tran.
exarninatior and 30%), ai hours, stain for observai
Individ was dissecte small amou:
and the styl each locule · fluorescence days to allo- A single middle and I in fluorescer also recorde
128
C. Field harvest and microscopy
A minimum of 10 pollinated flowers per treatment per tree were harvested at 1, 2, 3, 4,7, and 9 days after pollination in the November 2005 fieldwork, and at 4, 7, 9, and 14 days after pollination in the November 2006 fieldwork. The harvesting intervals were determined based on the results of previous studies of Myrtaceous species: Leptospermum sp. (O'Brien, 1994), E. globulus (Pound et al., 2002a), and E.
nitens (Pound et al., 2003). A minimum of 25 flowers subject to open-pollination were also collected on the 14'h day after they had opened during the November 2006 observations.
11
I:
II
B. PollinationMethods for controlled cross- and self-pollination used were as described by Baskorowati (2006). The treatment on the first experiment comprised two pollen treatments; pollen from the same tree (self-pollination) and pollen from different tree (cross-pollination). The treatments in the second experiment comprised of cross-pollination (32 x polymix, 48 x polymix, and 53 x polymix), self-pollination (32 x 32, 48 x 48, and 53 x 53) and open-pollination (each of 32, 48, and 53). In this experiment, a polymix comprising a mixture of pollen from 4 unrelated families collected during the November 2005 flowering season was used. The pollen mix, comprising pollen from family numbers 12, 15, 50, and 99 was 12 months old, and had been stored in a deep freezer at -l8°C. A pollen viability test undertaken before pollination, it was found to have a mean germination percentage of 49%. Pollen viability was tested using liquid medium in-vitro techniques (Griffin et al., 1982).
Pollen was incubated at 25°C in a solution of 30% sucrose to which was added 150 ppm of boric acid. Approximately 0.2 ml of the solution was transferred into a vial, and then a small amount of pollen picked up using a toothpick, was added to the vial. The vials were shaken, labelled and incubated at 25°C. Germination was recorded after 24 hours by counting the pollen grains under a microscope at 40X magnification. A minimum of 50 flowers per treatment per tree were emasculated, pollinated and bagged. Each flower of M. alternifolia consists of four petals, four sepals, and five staminal columns from which numerous anthers are attached by "short filaments, a single style and stigma; each flower has three carpels with numerous ovules (Baskorowati, 2008).
Research School of Biological Sciences (RSBS), Australian National University, Canberra. Pollen tube observation was carried out twice due to the number of flowers limitation during the flowering season.
The first experiment was undertaken in November 2005. A reciprocal mating design was used in this study: one individual of each of families 32 and 99 was crossed in each direction with an individual of the other, and both individuals were self-pollinated. However, due to limitation of flower available on those trees, families (32, 48, and 53) were used in the second experiment (November 2006).
Journal of Forestry Research Vol. 6 No. 2, 2009: 126-138
--- --
' . .
·-~
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-~'"~ . ····~
• • ;f• e=2~:l. ;" --- ... ~....;
129 A. First Experiment
The maximum number of tubes at the three positions (top, middle, and bottom) of style in the first experiment (November 2005) was recorded 4 days after pollination. Pollen grains had germinated on the stigma of the styles harvested at 1, 2, and 3 days after pollination; however, only low numbers of pollen tubes III. RESULTS AND DISCUSSION
Yij = observation
µ, = overall mean pi = replicate effect 'J = treatment effect
£1J = residual
Th e model was: Yij
= µ + pi +-rj
+ sij D. Statistical AnalysisRestricted (or residual) maximum likelihood (REML) was used as a model for analyzing the data. This approach was used in preference to analysis of variance (ANOV A) as it is capable of handling unbalanced data. The numbers of pollen tubes were transformed into square roots to meet a normal distribution assumption.
The number of pollen tubes was set as response variate and seedlots, position of observation, days to harvesting to styles were set as fixed effects. All analyses were performed with the statistical package GenStat.
The style and hypanthium from each treatment were fixed in Carnoy's fixative (6 : 3 : 1 absolute ethanol : chloroform : glacial acetic acid) for a minimum of 24 hours, transferred to 75% ethanol, and stored in a deep freezer (-18°C). Prior to examination, the fixed material was dehydrated through an alcohol series (90%, 70%, and 30%), and then softened in 0.1 N sodium hydroxide at 60°C for a minimum of 5 hours, stained with decolorized aniline blue overnight (Martin, 1959), and prepared for observation under a microscope.
Individual styles were incised longitudinally and the skin of the hypanthium was dissected and incised longitudinally so that the ovules could be seen clearly. A small amount of PBS (phosphate buffered saline) solution was added to the slides, and the styles were then gently squashed onto microscopy slides. All ovules from each locule were mounted onto microscope slides. The slides were observed using fluorescence microscopy; these slides could be stored in a refrigerator for up to 3 days to allow for subsequent observation.
A single count of the number of pollen tubes in each style was made at the top, middle and bottom of the style using a counter, under a Zeiss Axiophot microscope in Huorescence mode. The total number of pollen tubes penetrating the ovules was also recorded.
Pollen Tubes Growth ... L. Baskorowati
harvested at ork, and at 4, he harvesting f Myrtaceous 002a), and E.
n-pollination e November described by d two pollen ram different comprised of If-pollination
d 53). In this lated families e pollen mix,
nths old, and rtaken before f 49%. Pollen et al., 1982).
as added 150 ferred into a was added to ruination was scope at :1-0X emasculated, r petals, four
are attached carpels with n those trees, ber 2006).
procal mating 2 and 99 was
I University, e number of
errc mer poll Figure 3. Avt.
tubi
4< 120
"'O
~ 100
t~ > >- ('II::;;
80 v ~ ..0 ;: ·- E
~.g 60
=§ '§
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'O ~
] ~ 20
E 0
"
z 0
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'"/.., =
<
0.001); I harvesting of · The number c stat = 98.6, d.of the style w:
Figure 2. The stan:
U) 180 '
U) c
Q) 0 150 '
.0 :;::;
.3 'iii c 0 120 '
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0 (") .91
0. Q; 2:- 90.
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"'O .0 E :::} Q) Cl (U 30
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130
Results for the pollination treatments show that the average number of pollen tubes over the 3 positions in the style at 7 days after pollination, varied between crosses in the treatments (Figure 2; Wald stat = 114.4, d.f. = 3, X, =
<
0.001), for both self-pollination and cross-pollination. The average number of pollen tubes following cross-pollination was greater than that from self-pollination.A C
Figure 1. Fluorescence micrographs of pollen tubes growth on pistil of M.
alternifolia, stained with decolourized aniline blue. A: 4 days after cross- pollination; B and C: 4 days after self-pollination
B
grew down the styles towards the ovary. Therefore, results of the pollen tube growth assessment described here focus on styles harvested at 4, 7, and 9 days after pollination. In general, pistils from all types of pollination observed at 4, 7, and 9 days following pollination showed straight pollen tubes located within pollen tube walls (Figure LA). However, abnormalities were evident in the styles and in the ovules of several self-pollinated pollen tubes. Abnormalities included disoriented pollen tubes, multiple small tube clusters, and abortion (Figure LA and LB). These abnormalities are often used as indicator in incompatibilities (O'Brien, 1994).
Journal of Forestry Research Vol. 6 No. 2, 2009: 126-138
131 Figure 4. Average number of pollen
tubes and associated standard errors of M. alternifolia at different positions on the style (November 2005) · Figure 3. Average number of pollen
tubes and associated standard error of M. alternifolia at increasing intervals from pollination (November 2005) rer cross-
ler of pollen
lied between
f
0.001), forpollen tubes [.
Days to harvesting of styles.
upper middle lower
Position at the style 100
90
.z 80 70
Jl 2 60 0 50
c; 0. 40
~ 30
-" a , 20
z 10
0 120
""C
~ 100
t;~
> "
.
:;; 80v c -" 3 ·- E
c 0 60
()";::!
~·g 0. 0. 40 c; .., j ~ 20
8 0
z , 0
4 days 7 days 9 days
The period elapsed from pollination to style harvesting significantly influenced the average number of pollen tubes in the style (Figure 3; Wald stat= 27.1, d.f. = 2,
x
=<
0.001); the average number of pollen tubes declined significantly as interval toharvesting of the style increased, from a maximum at the 4rh day after pollination.
The number of pollen tubes also varied with position in the style (Figure 4; Wald stat = 98.6, d.f. = 2,
x
=<
0.001); the mean number of pollen tubes in the middle of the style was greater than those in the upper and lower parts of the style.Figure 2. The average number of pollen tubes over 3 positions in style, and associated standard errors, for self- and cross-pollination treatments (November 2005)
pollen tube 180
9 days after (/) (/) c:
lt4, 7, and 9 1l
-
:Js
·- (/) 150l pollen tube c: Q) 0 a. 120 ls and in the =
a
C"') m ~ (]) 90I
disoriented ... > (/)0 0 .£; 60
I ~ -0
~ 1.B). These 1l ~ 30
l,
1994). E "' :J c: ~ Q) > 0I
"'
32x32 32x99 99x32 99x99seedlot sarrple
Pollen Tubes Growth ... L. Baskorowati
48
32 53 Sample tree
Table l. Aver;
open- Figure 6. Aver
tubes error mere polli
70
8) and generall}
(Pound et al., 2(
132
:,
Observations of the ovules using fluorescence microscope suggest that no ovules were penetrated by pollen tubes at 4, 7, and 9 days after pollination. Some penetration was recorded 14 days after pollination. Analysis of data from 14 days after pollination shows that the number of pollen tubes penetrating the ovules varied between pollination treatment (Table l; Wald stat= 53.5, d.f. = 2,
x
=<
0.001), with cross-pollination resulting in the highest average number of ovulespenetrated by pollen tubes, followed by open- and then self-pollination treatments.
Pollen tubes were observed to have penetrated ovules at the micropyle (Figure Figure 5. The average number of pollen tubes over 3 positions in style and associated
standard errors for self-, open- and cross-pollination treatments (November 2006)
32 open 32 self 32 cross 48 open 48 self 48 cross 53 open 53 self 53 cross controlled pollination seedlots
.!; 120
VJ VJ
Q) c:
100 ..c .Q
.2 ~
c: VJ 0 80 "
~ a.
0 a.
"'
:;; ~ z- 600 > 0 VJ
:;; -0 40
.0 Q)
E tn 20
::J "'
c: :;;
0
>
"'
B. Second Experiment
As described in Materials and Methods, the pollen used for cross-pollination in this experiment (November 2006) was a mix from trees in four different families.
The result shows that the average number of pollen tubes varied between type of cross (Figure 5; Wald stat= 427.8, d.f. = 8,
x
=<
0.001). There was a great variation in the average number of pollen tubes between self-, cross-, and open-pollination, with fewer pollen tubes resulting from self-pollination compared to cross- and open- pollination. Figure 5 shows that average number of pollen tubes varied between sample trees, which family number 32 showed more average number of pollen tubes compared to other families. The variations of number pollen tubes between sample trees indicate the variation of self-sterility between the three sample trees.As in the preceding experiment, the average number of pollen tubes detected in the style also declined with the time from pollination to style harvesting (Figure 6), but the difference was not statistically significant (Wald stat = 7.7, d.f. = 2,
x
= 0.052). There were significant differences in the number of pollen tubes with position on the style (Figure 7; Wald stat = 53.84, d.f. = 2,
x
=<
0.001); the number of pollen tubes in the middle of the style was greater than those in its upper and lower sections.Journal of Forestry Research Vol. 6 No. 2, 2009: 126-138
133 0.8
1.3 1.3 7.5
4.2 5.3 0.12
0.21 0.15 48
32
53
Self-pollinated Cross-pollinated Open-pollinated Number of penetrated ovules per flower (%)
Sample tree
Table 1. Average of ovule penetrated by pollen tubes from self-, cross-, and open-pollination, 14 days after pollination (November 2006)
Figure 7. Average number of pollen tubes and associated standard errors of M. alternifolia by position in the style (November 2006)
Position at the style
Figure 6. Average number of pollen tubes and associated standard errors of M. alternifolia at increasing intervals from pollination (November 2006)
lower middle
upper
7 9 14
Days to harvesting of styles
80 70
.D 60
3 50
0 .§ 0. 40
0 30
u 20
.D z E ~ 10
0
70
-0 60
*'
t~ 50
> "
" t;;
u " 40
.D ~
-
-- ~ " 30~:~ 0 0. 0. 0 20
or"'I
" " 10
~ ~
E 0
c 0
z
8) and generally had grown through the nucleus tissue towards the embryo sacs (Pound et al., 2002a).
Pollen Tubes Growth ... L. Baskorowati
uggest that no
~lination. Some fa from 14 days
ing the ovules d.f. = 2,
x
= mber of ovules ion treatments.ropyle (Figure and associated ts (November
53 self 53 }ross
!-pollination in [erent families.
etween type of
!great variation en-pollination,
~oss- and open- [aried between nber of pollen
!tubes between 'sample trees.
rtubes detected 1"esting (Figure
[.7,
d.f. = 2,x
llen tubes with ' <0.001); the
se in its upper
Less poller styles. Howeve this species. Tl pre-zygotic me IV. CONCU Abnormal growth, irregu in this study (l were found ir pollinations.
In this sn pollination die ovules being h preparing ovu specimen, may results of polle found in E. glo case, a more ex is suggested; er results.
Pollen tu ovules, but on is consistent v
Suggesting th in other WOO·
in M. alternifo suggests that , self incompat (1988) found 1 pollination; S1 self incornpati seed developrr (Hodgson, 19/
self-pollinatio:
self pollen tu!
(1987) and Ell in E. regnans, al. (2002a) rep 100% self-inco strong mechar al. (2002a) con mechanism.
134 /
The rate of pollen tube growth in M. alternifolia is slow compared to that of angiosperms (Heslop-Harrison, 1971). Results from 2005 observation shows that pollen tubes do not grow between the cells of the transmitting tissue in the centre of the style for 48 hours, and only reach the base of the style 4 days after pollination.
The results reported here are, however, similar to those reported for other member of the Myrtaceae, such as E. regnans, Leptospermum myrsinoides, L. continentale, Chamelaucium uncinatum and Metrosideros excelsa (Griffin et al., 1987; Sedgley et al,. 1989; O'Brien and Calder, 1993; O'Brien, 1996; Schmidt-Adam et al., 1999).
The reduction in number of pollen tubes from the upper part to the lower part of the style, i.e. from the stigma to the ovaries, suggests some selection between pollen tubes is occurring in the style. This may result from competition for space
to penetrate the stigma and enter the style, or competition between the fastest growing tubes to reach the loculus and the ovaries (Ellis and Sedgley, 1992; O'Brien, 1996). Moreover, pollen presenter and upper style are recognized as the control points in pollen tube growth (Fuss and Sedgley, 1991). The pollen presenter has a different morphology from the rest of the style, with an abundance of transfer cells surrounding the transmitting tissue (Clifford and Sedgley, 1993). The upper style is the next controlling region, with smaller numbers of pollen tubes inhibited in the pollen presenter (Maguire and Sedgley, 1998). The results from two different periods of observation demonstrate that the number of pollen tubes following self-pollination was less than that from cross-pollination and open-pollination;
this result also showed that rejection of self-pollen tubes occurs in the style as a mechanism of self-incompatibility. Rejection in the stigma or the style is a common self-incompatibility mechanism in plants (de Nettacourt, 1977; Lewis, 1979), which was demonstrated in E. woodwardii flowers (Sedgley and Smith, 1989). The results of this study, that M. alternifolia flowers are preferentially outcrossing, confirms the result of a previous study based on outcrossing rate (Butcher et al., 1992).
Figure 8. Fluorescence micrograph of arrested pollen tube in the ovule 2 weeks after cross-pollination, the hypanthium was stained with aniline blue (November 2006)
Journal of Forestry Research Vol. 6 No. 2, 2009: 126-138
135 Less pollen tube was found in the self-pollinated styles than cross-pollinated styles. However, the result shows that mechanism of self-incompatibility occurs in -this species. The rejection of self-pollen tubes at the ovarian level confirmed that
pre-zygotic incompatibility system occurred in M. alternifolia.
IV. CONCLUSION
Abnormality of pollen tubes in self-pollinated crosses, such as disorientation of growth, irregular tubes and spiraling tubes in the self-pollination style, was evident in this study (Figure 1.B and 1.C). A higher number of pollen tube abnormalities were found in self-pollinated flowers than those subjected to cross- and open- pollinations.
In this study, more than 50% of ovules samples observed 2 weeks after pollination did not show penetration by a pollen tube. This probably due to the ovules being harvested too early, prior to fertilization; or the physical action of preparing ovules for microscopic examination, which involved squashing the specimen, may have caused pollen tubes to be pulled out of the micropyles. Unclear results of pollen tubes in the ovules due to the inappropriate methods were also found in E. globulus (Pound et al., 2002b) and E. nitens (Pound et al., 2003). In this case, a more extended time for observing fertilization of the ovules of M. alternifolia is suggested; embryological studies should be undertaken to help provide clearer results.
Pollen tubes resulting from self-pollination are also capable of penetrating the ovules, but only a few do so compared to cross- and open-pollination (Table 1). This is consistent with the results reported in E. woodwardii (Sedgley and Smith, 1989).
Suggesting that a late-acting self-incompatibility mechanism, such as that found in other woody species (Bawa et al., 1985; Seavy and Bawa, 1986), is also active in M. alternifolia. In conjunction with the rejection of pollen tubes in the style, it suggests that M. alternifolia has multiple barriers to self-pollination. The level of self incompatibility varies among the Myrtaceae. For example, Potts and Savva (1988) found complete sterility in E. morrisbyi with no seed production after self pollination; Sedgley and Smith (1989) reported that E. woodwardii showed partial self incompatibility since self-pollination resulted in reduced capsule retention and seed development as compared with cross-pollination; this also occurs in E. grandis (Hodgson, 1976). Sedgley and Smith (1989) concluded that the lack of seed set from self-pollination in E. woodwardii was due to a lack of penetration of ovules by the self pollen tubes rather than a post-zygotic mechanism. However, Griffin et al.
(1987) and Ellis and Sedgley (1992) described a post-zygotic mechanism operating in E. regnans, as well as in E. spathulata, E. cladocalyx and E. leptophylla. Pound et al. (2002a) reported that four of five trees of E. globulus spp. globulus were either 100% self-incompatible or greater than 99% self-incompatible, indicating that a very strong mechanism operated to prevent selfed seed from being produced. Pound et al. (2002a) concluded that self incompatibility within Eucalyptus can be a complex mechanism.
Pollen Tubes Growth ... L. Baskorowati
~red to that of bn shows that
I
n the centre ofl
er pollination.
other member '· continentale, t87; Sedgley et eta!., 1999).
the lower part ction between ition for space een the f ~stest 1992; O'Brien, as the control presenter has a ef I transfer cells [he upper style
1es inhibited in
1 two different
~bes following
I 11· .
en-po matron;
~ the style as a
I •
~e rs a common
~, 1979), which 9). The results g, confirms the 1992).
[ 2 weeks after lue (November
~1 .. ,_
Journal of Forestry Research Vol. 6 No. 2, 2009: 126-138
ACKNOWLEDGEMENTS Frankli
The author is very grateful to Prof. Peter Kanowski, Dr.John Doran, Mr. Mike 1D Moncur for their invaluable comments and supports. Thanks are also due to Dr. Sc Sally Stowe and Dr. Cheng Huang for fluorescence microscopy assistance and Dr. Fuss, A
Emlyn Williams for his support in statistical analysis. R
Griffin
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~er Verlag, New lers for breeding
Id
Development g system of three blian Journal ofic diversity
. .
and ceae). Australianf
1 the Rubiaceae 70: 1281-1288.
Grayum. 1985.
s: II Pollination ical community.
feuca alternifolia
f
IAR, Canberra.ralia (Maiden &
chool Resource sity, Canberra, laleuca. In: E.G.
The School of
1 oran, Mr. Mike also due to Dr.