UNIVERSITI PUTRA MALAYSIA IN VITRO CULTURE AND SYMBIOTIC RELATIONSHIPS OF PAPHIOPEDILUM NIVEUM, TAENIOPHYLLUM OBTUSUM AND VANDA HOOKERIANA WITH THEIR ASSOCIATED FUNGI IRAWATI FP 1987 8 It is hereby cert i f ied that we have read th is thesis ent itled "In V itro Culture and Symb iotic Relat ionsh ips o f Paphiopedilum niveum, Taeniophyllum obtusum and Vanda hookeriana w ith the ir Assoc iated Fung i" by Irawati, and in our op in ion it is satis factory in terms o f scope, qual ity, and presentation as partial ful f ilment o f the requ irements for the degree o f Doctor o f Ph ilosophv . .-:-�.':'�:�: . SULAlMAN M. YASSIN, '":") � Ph.D. Professor Dean of Graduate Studies Un ivers it i Pertanian Malaysia (Cha irman Board of Exam iners) � r\ I) 1 � .. ..... ......... �jt/L rLtll- \\ JOSE H AR ITTI, Ph.D. Pro fessor School o f B iolog ical Sc iences Univers ity o f Cali forn ia, U.S.A (External Exam iner) Jg.<� i:t,. � GioF�Y . HABL�Y : . Ph: ( C-'l D. --- Department o f Plant Sc ience University o f Aberdeen St. Machar Dr ive, Aberdeen England (External Examiner) MOHD. ZAIN HJ. ABD. KARIM, Ph.D. Pro fessor/Dean Faculty o f Agr iculture Un ivers iti Pertanian Malaysia (Internal Examiner) MOHAMMAD MD. AL I, Ph.D. Assoc iate Pro fessor Faculty o f Agriculture Univers iti Pertan ian Malays ia ( Internal Examiner/Superv isor) This thesis was submitted to the Senate o f Universiti Pertanian Malaysia and was accepted as partial fulfilment of the requirements for the Degree o f Doctor o f Philosophy. Date: .. ,l i J U L t:)�� SULAIMAN M. YASSIN, Ph. D. Pro fessor Dean of Graduate Studies IN VITRO C UL TURE AND SY MBIOTIC RELATIONSHIPS OF PAPHIOPEDILUM NIVEUM, TAENIOPHYLLUM OBTUSUM AND VANDA HOO KERIANA WITH THEIR ASSOCIATED FUNGI by Irawati A thesis submitted in partial fulfilment o f the requirements for the degree o f Doctor o f Philosophy in the Faculty o f Agriculture, Universiti Pertanian Malaysia September 1987 AC KNOWLEDGEMENT To my individuals and institutions I would l ike to express appreciation and sincere gratitute for their contributions in completion of this study . * My supervi sors , Pro fessor ) and Dr . Mohammad Dr . Hasan Mad Md . Ali ( Associate for their guidance and advi ce throughout the course of this study and in the preparation of this manuscript . * Mr . Saleh Kadzimin , for all his help since the beginning of this study . * Mr . Mark A . Clements , for the valuable discussions , his helpful comments and suggestions and also for his help in fungal determination . * Dr . Zaliha Christine Alang , for reading thi s thesis . * The of Faculty the of Agriculture , and the Universiti facilities in Pertanian conducting this Computer Centre Malaysia , research for and the in preparing this manuscript . * Co mmonwealth �ycological identification . ii Institute for fungal * Mr Ho O i Kuan, Mrs. Am inah, Mr Mr Yusof Suki, How Yee Peng, Prof. Yap Thoo Chai for Ms. Fadzlon Mohd. Juso f, Mr Mimbar Haryadi, Dr. La i Chao May and their assistance and also to Mrs. Chairun Nisa Haris and Mrs. Kamariah Hamid for the ir kind help. * Public of a Services fellowship Department o f Malaysia for the award under Assistance Programme. iii the Malays ian Technical TABLE OF CONTENTS Page ACKNO WLEDGEMENT . . TABLE OF CONfEN'I'S LIS T OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • • . . . . . • • . . • • • • • . . • . • . . • . . • . . • . . • • • • • . . . . . . . . . . . . • . ..................... LIS T OF PLATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LIS T OF ABBREVIA TIONS • • • • . • • • . . • • • • . • • • • • . • . • • • • . • • • . . • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • CHAPTER I I . xii xvi xvi i 1 INTRODUCTION CHAPTER I . ix xv LIS T OF APPENDICES • iv xi LIS T OF FIGURES ABS TRACT ii 6 LI TERATURE REVIEW 8 A. Plant Materials B. Tissue Culture of Orchids 13 C. Symbiotic Germination of Orchid Seeds 17 D. Fungal Myc orrhiza Associated with Orchids 23 E. Symbiotic Development of Germinated Seeds 29 F. Asymbiotic Germination of Orchid Seeds 34 G. Establishment of Seedlings 37 CHAPTER III. A. . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 40 40 Plant Materials 1. . Paphiopedilum niveum Rchb . f . 40 a. In vitro culture 40 b. Seed 41 c. Protocorm-l ike b ody 42 iv Seedling e. Fungus ................. .... .. 42 ..... ........ ........... . 42 . . ........... .. . ....... .... a. In vitro culture b. Seed c. Protocorm-like body d. Seedling e. Fungus . 3. . . .. . . ......... ..... .............. . 43 44 44 45 .. .... ......... ....... 45 ......................... . 46 . . . . . . . 45 a. In vitro culture 47 b. Seed 47 c. Protocorm-like body 47 d. Seedling 48 e. Fungus 48 .... ......................... . 49 Media 2. . Yanda hookeriana Rchb . f . 3. 1. . . . Taeniophyllum obtusum Bl . 2. B. d. Paphiopedilum n iveum Rchb . f . 50 a. In vitro culture 50 b. Seedling 51 Taeniophyllum obtusum Bl . 52 a. In v itro culture 52 b. Protocorm-like body 54 c. Seedling 55 Yanda hookeriana Rchb . f . 56 a. In vitro culture 56 b. Protocorm-like body 56 c. Seedling 57 v c. ............................... 57 ...................... 58 • 59 4. Fungus 5. Symbiotic studies . . a. Seed stage b. Protocor m-l ike b ody stage 59 c. Seedling stage 60 • • • • • • • • • • • • • • • • • • • • • • • 60 Techniq ue/Working Methods 1. 2. 60 In vi tro culture 60 a. Paphiopedilu m niveu m Rchb . f . b. Taeniophyllum obtusu m Bl . 63 c. Yanda hookeriana Rchb . f . 65 Preparation of seedling . • • • • • • • • • • • • • • • • • • • • • . • . . • • • • 66 66 a. Paphiopedilu m niveu m Rchb . f b. Taeniophyllu m obtusu m Bl . 66 c. Yanda hookeriana Rchb . f . 67 • 3. Isolation of fungi 68 4. Methods of inoculation 70 a. Seed s tage 70 b. Protocor m-l ike bod y stage 72 c. Seedling stage 73 5. Environmental conditions 74 6. Description of treatments 7. • • • • • . • • • • • • • • a. Seed stage b. Protocor m-like bod y stage 76 c. Seedling stage 78 Observation a. . ........................ 74 .... ............ .... .... . In vitro culture vi . . 74 79 79 b. Symbiotic germination c. Symbio tic development of protocorm- like b o dy . ... ......... . . .. ............ .... 80 d. Symbiotic development of see dling . . 80 e. Observation under light microscope . 80 f. Observation . . . . . electron un der 83 microscope RESULTS 85 In Vitro Culture 86 C HAPTER IV. A. 79 1. Paphiope dilum niveum Rchb . f . 2. Taeniophyllum obtusum Bl . 3. • • • . • • • • • • • • • • • • • • • . • • • • • 86 89 a. Root culture 92 b. Infloresc ence culture 95 c. Shoot tip culture 96 Van da hookeriana Rchb . f . 99 B. Fungal Culture 101 C. See dl ing 121 D. 1. Paphiope dilum niveum Rchb . f . 1 21 2. Taeniophyllum obtusum Bl . 1 28 3. Yan da hookeriana Rchb . f. 1 35 Symbiotic Germination 1 36 1. Paphiope dilum niveum Rchb . f . 2. Taeniophyllum obtusum Bl . 3. Yan da hookeriana Rchb . f . vii 1 37 ........ . . .... ..... ... .... . . . 1 40 1 48 E. F. Symbiotic Development of Protocorm-like body . 161 1. Taeniophyllum obtusum Bl . 162 2. Vanda hookeriana Rchb . f . 1 66 Symbiotic Development o f Seedling 1 69 1. Taeniophyllum 2. Vanda hookeriana Rchb . f . 1 69 obtusum Bl . 175 DISCUS SION 1 80 A. I n vitro culture 180 B. Fungal culture c. Seedling 185 D. Symbiotic germination 191 E. Symbiotic development of protocorm-like body . 197 F. Symbiotic development o f seedling 1 99 CHAPTER V . CHAPTER VI . CHAPTER VII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 SU MMARY AND CONCLUSIONS BIBLIOGRAPHY . . . . . . . . 1 83 . . . . . . . . . . . . . . . . . . . 209 222 APPENDICES viii LIST OF TABLES page Table 1. Envi ronmental conditions in various trials 75 2. Treat ments o f the seed 77 3. Treatments o f the protocor m-like body 78 4. Dec ontamination niveu m explants procedures for Paphiopedilum 88 5. Deconta mination procedures for obtusu m explants Taeniophyllum 6. Taen iophyllu m obtusu m root tip cultures on modified Knudson's C mediu m at different pH after two weeks . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 The effect of sterile coconut water or hor mones added to Taeniophyllu m obtusu m root tip cultures after two months 94 Dark treat ment on Taeniophyllum obtusu m root tip cuI tures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Ef fect of BA or NAA on inflorescence cultures of Taeniophyllum obtusu m 96 Fungi isolated from Paphiopedilum niveu m, Taenioph yllu m obtusu m and Vanda hookeriana at dif fer ent ti mes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 11. Duration ( days) for the fungal hyphae to reach one c enti meter in Fungi isolating mediu m 118 12. The growth of P I , T l and VI fungi on different media after six days in culture 119 Develop ment of fungi fr om three different orch ids on Oat mediu m and fungus P I on mixtures of Oat mediu m 1 20 Ger mination of Paphiopedilum niveu m seeds at different maturity 124 Germination of Taeniophyllum obtusu m seeds at different medi a . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 29 . 7. • 8. • • • • • • • • . . . • • • . 9. . 10. . • • • • • • • 13. • 14. . 15. . . . • . . • . • . • . • . . . • . • • • . • • . . • • • . • • • • . • • . • • . . . • • . • • • . . • • . • • • . • . • . • • • . • • • . . ix 91 page Tab le 16. Ger mination of Taenioph yllum obtusu m seeds at different maturity . . . . . . . . . . . . . . . . . . . . . . . . . . 17. Paphiopedilum niveu m different treat ments 18. Ti me of development of part s during the germination of Taeniophyllu m obtusu m seeds 19 . Taeniophyl lu m obtusu m different treatments 20 . Ti me of develop ment of parts during germination of Vanda hookeriana seeds seed germination seed at germination • seed at the • • • germination • • 130 1 39 1 46 1 47 152 21 . Mean Vanda hookeriana different treatments 22 . Average number of Vanda hookeriana leaves and roots per seedling . . . . . . . . . . . . . . . . . . . . . 158 23 . Develop ment of Taeniophyllum obtusu m protocor m -like bodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 24 . Develop ment l ike bodies 167 25 . Averag e number of leaves and roots protocor m-like body of Vanda hookeriana 26. Protocor m bud ding seedlings 27. Survival seedlings at . 28 . of Vanda hookeriana . . .... . . Additional weight obtusu m seedling . . Taeniophylum obtusu m of . . • . . . . . ....... . .. . . . • . . . . .... . gra m Taeniophyllu m per . per Taenioph yllu m obtusu m in ability protocor m- . . . . . . . . . . . . . . . . . . • . . 1 53 168 1 70 172 1 74 29 . Nitrog en , phosphorus , potassiu m, iron and zinc content of Taenioph vllum obtusum seedlings 176 30 . Survival ability of Vanda hookeriana seedlings 177 31 . Nitrogen , phosphorus , potassiu m, iron and zinc c ontent of Vanda hookeriana seedlings 1 79 • x • • • • • . LIST OF FIGURES Figure 1. 2. page Correlation between rate of germination and perc entage of germination of Paphiopedilum niveum seeds 125 Correlation between rate of germination and age of Paphiopedilum niveum seeds 126 Correlation between percentage o f germination and age of Paphiopedilum niveum seeds 1 27 Correlat ion between rat e of germination and perc entage of germination of Taeniophyllum obtusum seeds 132 Correlation bet\veen rate of germination age of Taeniophyllum obtusum seeds 133 • 3. • • • • • 4. . 5. . • • . • . . • • . . • • . • • • • • • • • • • • • • • • • • • • • • • • . • • • • . • • . • • . and • • • 6. Correlation between perc entage of germination and age of Taeniophyllum obtusum seeds 1 34 7. l eaf number per seedling Mean of Yanda hookeriana at dif ferent treatments ( Tl , VI, V4 , KCbc ) 1 57 root number per seedling Mean of Yanda hookeriana at different treatments ( Tl , VI, V4 , KCbc ) 1 59 8. xi LIST OF PLATES PLATE I. II. III . IV . page 9 Paphiopedilum niveum flower Taeniophyllum obtusum 11 Yanda hookeriana 12 Young flower b ud of Paphiopedilum after seven months in culture • V. VI . • • • • • • niveum • • • • • • • 90 • 90 Shoot tip cul ture of Paphiopedillum niveum Section of Taeniophyllum obtusum inflorescence culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 . VII . VII I . obtusum Multiple branches of Taeniophyllum infl orescence culture ( NAA treatment ) Roots developing from Taeniophyllum obtusum inflorescence culture ( BA treatment ) 100 Yanda hookeriana protocorm-like bodies 100 • IX. X. Fungal root di stribution XI . Fungal root distribution in XII . XIII . • • • • • • • Scanning electron microgram of digested fungal clumps in Paphiopedilum niveum roo t Digested fungal clump root Living root . . . . . . . fungal . . . . . . . • • • • • • • • in Paphiopedi lum niveum . . 102 Taeniophyllum obtusum Fungal distribut ion in Vanda hookeriana root . XV . • Paphiopedilum niveum in • XIV . 97 . . . . . . . . . . . . . . . . . . . . . . . clumps in Paphiopedilum niveum 102 . 103 104 104 105 XVI . Thick fungus cell wall inside the host cell . . 1 07 XVII . Thin fungus cel l wall ins ide the host cell . . . 107 Thick cel l wall of digested hyphae 108 XVI II . xii PLATE XIX . XX . page Cellulose layers at both s ides of host cell wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Transmission clump 1 10 . . . . . electron . . . . . . . . . . microgram . . . . . . . . . . . . of a fungal .... . . . . . . . • XXla . Fungus PI from Paphiopedilum n iveum 1 14 XXlb . Fungus P2 from Paphiopedilum niveum 114 XXlc . Fungus P3 from Paphiopedilum niveum 114 XXld . Fungus P4 from Paphiopedilum niveum 1 15 XXle . Fung us T l from Taeniophyl1um obt usum 1 15 XXlf . Fung us VI from Yanda hookeriana 1 15 XXlg . Fungus V2 from Yanda hookeriana 1 16 XXlh . Fungus V3 from Yanda hookeriana 1 16 XXIi . Fungus V4 from Yanda hookeriana 1 16 XXlla . Paphiopedilum n iveum seed after Swollen six weeks in culture . . . . . . . . . . . . . . . . . . . . . . . . 141 XXllb . contamination Bacterial Paphiopedilum niveum seed 14 1 XXIII . Paphiopedilum n iveum protocorm grown from seed inoculated with PI (Tulasnella sp . ) fungus after three months in culture 142 obtusum protocorm grown from Taeniophyllum seed inoculated with Tl (Ceratobasidium sp . ) fungus after 20 weeks in culture 145 obtusum seed Culture of Taeniophyllum inoculated with Tl (Ceratobasidium sp . ) fungus after one week in culture 149 Bacterial contaminations on Taeniophyllum seeds inoculated with Tl obtu sum ( Ceratobasidium sp . ) fungus after one week in culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 49 germinating on • XXIV . • • • • XXV . • XXVI . xiii • • • • • • • • • • • • • • • • • • . • • • • • • • • • . • • • • PLATE XXVII . XXVIII . XXI X . xxx. page Multiple branche s of Taeniophyllum obtusum protocorms in complete medium (modified Knudson ' s C medium + bean sprout extract ) 1 50 Fungus VI (Ceratobasidium s p . ) germinating Vanda hookeriana seed 156 Protocorm-like bodies of obtusum in simple (Oat ) medium covered the Taeniophyllum Protocorm-like bodies of Taeniophyllum obtusum in modified Knudson ' s C medium 16 5 Protocorm seedling 1 71 . XXXI . 165 budding of x iv . • • . • Taeniophyllum obtusum LIST OF APPENDICES APPENDIX page A Modified Vacin & Went medium B Modified Knudson ' s C medium (Arditti , 1974) C Oat medium (Clements , 198 1 ) 222 • • • • • • • • • • Fungi isolating medium (Clements , 1981 ) xv • • • • • • • • • 223 224 224 LIST OF ABBREVIATIONS BA 6-benzyladenine 2,4-D 2,4-dichlorophenoxyacetic acid EDTA ethylenediamine tetraacetic acid FAA formol acetic alcohol IAA indole-3-acetic acid NAA naphthaleneacetic acid ppm parts per million TBA tertiary butyl alcohol xvi An abstract of the thesis presented to the Senate o f Universiti Pertanian Halaysia i n partial ful filment o f the requirements for the Degree o f Doctor o f Philosophy. IN VITRO CULTURE fu�D SYMBIOTIC RELA TIONSHIPS OF PAPHIOPEDILUI'1 NIVEUM, TAENIOPHYLLUM OBTUSUM AND VANDA HOOKERIANA WITH THEIR ASSOCIA TED FUNGI by Irawati September, 1 987 Supervisor Dr. Mohammad Hd. Ali Co-supervisor Dr. Hasan Mad Faculty Agriculture In vitro Paphiopedilum cultures of three orchid niveum, Taeniophyllum species, obtusum and namely, Yanda hookeriana, were initiated from dif ferent parts o f the plants. Proliferous tips protocorm-like bodies were obtained and axillary in florescence o f P. niveum V. hookeriana T. obtusum. grew In florescence branches buds o f into culture o f shoot and shoot tips and Shoot tips and axillary buds o f single T. o f in florescences, from or mUltiple plantlets. obtusum developed into multiple calli or produced roots. Young lea f tip or leaf base o f P. niveum and root o f T. obtusum were unable to dif ferentiate into young flower bud o f P. niveum callus. produced months. xvii However, leaf culture of a fter seven High pH of t he medium was suitable for Low concentration of modified preferable to the standard T . obtusum Knudson's concentration for C cultures . medium was the culture of P. niveum . Efforts to raise seedlings of the three di fferent orchids 140 to 160 -day showed that seeds n iveum and 60 to lIS-day old fruits of better than dry from seeds , o ld fruits of P. T . obtusum germinated while for V. hookeriana dry seeds were able to germinate freely . Myc orrhizal fungi isolated f rom P . niveum , T . obtusum and V. hookeriana their own roots were able to induce the Fungal mycorrhiza from seeds . germination T . obtusum showed a greater abi lity to initiate germination and promote growth V. hookeriana Other fungi deleterious Most not seeds fungi influenc e and species were seeds . associated the of own fungal mycorrhizas . associated with the three orchids to the V. hookeriana compared to i t s of with these three development of protocorm-like were not compatib le to orchids did bodies T. obtusum . of Their influence in the establishment of V. hookeriana and T . obtusum seedlings in the nursery was also not s ignificantly shown in this study . xviii Abstrak tesis yang dikemukakan kepada Senat Universiti daripada Pertanian Malaysia sebagai memenuhi sebahagian keperluan untuk ijazah Doktor Falsafah. KULTUR I� VITRO Dfu� P ERHUBUNG AN SIMBIOSIS ANTARA PAPHIOPEDILUM NIV EUM, T AENIOPHY LLUM OBTUSUM DAN VANDA HOOKERIANA D ENGAN KUL AT-KUL AT SEKUTUANNY A oleh Irawati September, 1987 Penyelia Dr. Mohammad Md. Ali Penyelia bersama Dr. Hasan Mad Fakulti Pertanian Kultur Paphiopedilum hookeriana, in vitro niveum, telah Struktur-struktur cepat bagi spesies Taeniophyllum dibuat dari diperolehi dari pucuk samping dari P. niveum Kultur jambak dan bahagian Pucuk dan tunas T. obtusum. tumbuh menjadi anak benih tunggal atau bunga dari T. obtusum tumbuh ganda, kalus atau menghasilkan akar. pangkal daun yang muda dari muda P. niveum dan akar kalus. P. niveum tanaman. V. hookeriana jambak bunga dapat menghasilkan Vanda yang mampu �embiak dengan dan tunas samping bunga iaitu, orkid, obtusum beberapa seperti protokom dan dari pucuk dan jambak ganda. tiga dapat bulan. xix daun Hujung dan T. obtusum tidak Akan tetapi kultur menumbuhkan menjadi kuncup bunga setelah tujuh Medium yang T. obtusum. mempunyai pH Kepekatan tinggi adalah yang rendah sesuai untuk kultur dari medium Knudson's C yang diubahsuaikan adalah lebih baik bagi membiakkan P. niveum daripada yang berkepekatan biasa. Usaha untuk menumbuhkan anak benih dari ketiga-tiga jenis orkid yang berbeza didapati biji benih 140 hingga 160 hari dan biji benih T. obtusum yang berumur 60 daripada biji keringnya, hingga 115 hari bercambah lebih baik manakala biji kering P. niveum yang berumur V. hookeriana pula bercambah dengan bebas. Kulat-kulat mikoriza P. niveum, T. obtusum dan percambahan T. obtusum biji baik orkidnya. menunjukkan keupayaan dibandingkan Kulat-kulat V. hookeriana benih menggalakkan pertumbuhan lain dari mampu Kulat memulakan biji-biji dengan yang diasingkan yang merangsangkan mikoriza dari percambahan dan V. hookeriana kulat-kulat akar-akar yang lebih mikorizanya bersekutu dengan ketiga sendiri. jenis orkid merosakkan biji-biji benihnya. Kebanyrucan jenis orkid ini kulat-kulat yang bersekutu dengan ketiga-tiga tidak mempengaruhi pertumbuhan struktur struktur seperti protokom dari V. hookeriana dan tidak bagi T. obtusum. anak benih sesuai Pengaruhnya terhadap penyesuaian tumbuh dari V. hookeriana dan T. obtusum pada semaian benih juga tidak tampak berkesan dalam penyelidikan ini. xx anak CHAPTER I I N T Among ornamental U C T ION R OD plants, orchids are one economically important in this region. made to support the propagation practices seedlings, are marketing plants over-collection valuable techniques of to ful fil through cultivation the demand However, recently (Hagsater world have germplasms from are e f forts are methods, ,dId orchids or land measures Conservation studies most of people possible loss o f many orchid species due to the in countries breeding or cut flowers. aware o f the There fore the development o f orchid industry techniques, and of to be clearing & Steward, taken to extinction. in 1986 ) . prevent Suitable needed to multiply them. many This these propagation e f fort also encourage further breeding and utilization o f the would orchid species and also has a signi ficant role on their conservation. There them, three they are are species but is had Malaysian been orchids, and among chosen for this Paphiopedilum niveum, Yanda hookeriana. in nature. many important Taeniophyl1um research and obtusum Paphiopedilum niveum is restrictedly and found This species is not only an attractive wild orchid also a potential species for breeding. 1 Its commercial
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