Studies on Propagation of Tissue Culture Seedlings in Mulberry (Morus alba var. Shidareguwa)

Abstract

''Shidareguwa'' mulberry (Morus alba var. Shidareguwa), a synonym of ''Pendulate'' mulberry (Morus alba var. Pendula), is well known and popular for landscaping, gardening and fresh purposes because of its weeping canopy and berry production. However, this variety is difficult to propagate and its cuttings do not root well by conventional asexual methods. Thus, tissue culture might be typically the best method for the rapid propagation. Despite shoot induction through organogenesis from immature leaf explants in vitro has been reported in ''Shidareguwa'', however, further rooting induction of excised young shoots from regenerated ones were not obtained. On the other hand, so far, no substantial plantlets production system through micropropagation has been available in this species yet. The aim of current study was to establish a tissue culture method for plantlet production in ''Shidareguwa'' mulberry. Healthy and young growing shoot tips along with nodal explants were collected from 3-year-old mature trees grown at Danshui, New Taipei City, Taiwan; the collected materials were sterilized and subsequently cultured in various concentrations of MS medium. The effects of cytokinins (BAP, TDZ and coconut water) on shoot multiplication and regenerated shoot growth and the effects of auxins (IBA, NAA and 2,4-D), explant types and the times required on root induction and growth were examined in vitro and ex vitro, respectively. Moreover, the survival rate of plantlets was identified after the plantlets had undergone 1 to 2 months of a two-step process of acclimation, i.e., first in a climate-controlled room and then in a greenhouse. Finally, the success of transferring plantlets into the field was examined. A high rate of elimination of contaminants was found in explants soaked in 0.3 Clorox for 1 night before sterilization methods. A greater survival percentage was obtained when the shoot tip explants were used in the initial cultures based on MS medium containing 2% (w/v) sucrose and 0.8% (w/v) agar under room temperature at 25℃ and a light intensity of 56 μmol m-2 s-1 for one month. Basal MS medium supplemented with 4.44 μM BAP in initial culture and shoot multiplication showed that this composition induced the optimal growth of shoots. In medium and controlled environmental conditions described above, shoot length reached 9.3 mm with a maximum of 3 leaves per shoot tip after 20 days in the initial culture. Subsequently, after 1 month of incubation and shoot multiplication cultured on MS medium containing 3% sucrose and supplemented with 4.44 μM BAP, 4.1 shoots were produced per explant, with a length of 2.1 cm and 5.3 leaves. Young shoots excised from regenerated shoots were well rooted (90%), with the highest root number, i.e., 4.8-6.8 roots per explant with 3.3-5.7 cm of root length, cultured in MS medium containing 3% sucrose but lacking auxins after 30 days in vitro. In addition, 100% of root induction was observed when 3-4 leaves of shoots excised from regenerated shoots were cultured in pots containing perlite:vermiculite:peat moss in a 1:1:1 ratio with/without IBA ex vitro. However, shoots dipped in 2000 ppm IBA powder exhibited the greatest root number (8.3) per shoot and 2.2 cm of root length. The well-rooted plantlets obtained from in vitro experiments were subsequently transferred to plastic pots containing the same media described above and kept in an office room for one month of initial acclimation, leading to a survival percentage of 94%. Regardless of whether the plantlets were rooted in vitro or ex vitro, the survival percentage reached 100% when the plantlets experienced one months of secondary acclimation in a greenhouse equipped with intermittent mist and overhead shade. At last, a very high rate, i.e. 92% of success was acquired once theses plantlets were transferred into the field after the following 4 weeks. We concluded that an effective micropropagation of plantlet production system for ''Shidareguwa'' mulberry using shoot tips from mature trees through initial culture, shoot multiplication, root induction and acclimation has thus been successfully established.‘枝垂桑’ (''Shidareguwa'' mulberry)【(Morus alba var. Shidareguwa；別名（synonym）''Pendulate'' mulberry (Morus alba var. Pendula)】因其枝條下垂 (weeping)、樹姿優美並可生產果實，而為世界著名的庭園及景觀等觀賞樹木，並可為果樹之用。然而，本物種（species）極難以扦插、高壓等傳統無性繁殖方法大量而迅速地增殖，組織培養可能是解決此一問題的最佳方法。惟迄今，該種桑樹於微體繁殖(micropropagation) 的研究進展有限，僅止於以葉片為培植體（explant），經器官發生 (organogenesis) 而產生枝條 (shoot)，未有進一步成功誘導根系的發生，而產生完整的瓶苗並除瓶定植至田間的文獻發表。 為建立枝垂桑組培苗之繁殖系統，以新北市淡水區之3年生植株為採穗母株，取含健康莖頂（shoot tip）及其下數節（nodal segments）之新梢為培植體。採集之培植體首先經測試消毒與培養基條件及所須時間後發展初代培養（initial culture）之最佳的培植體型式（explant type），隨後決定繼代培養（subculture）枝條再生（shoot multiplication）之最適培養基，繼而探討瓶內（in vitro）與瓶外(ex vitro)發根之最佳培植體型式與條件，隨後經瓶苗出瓶、室內及溫室馴化，最終定植於田間，並分別計算其成活率，俾建立其組培苗之大量生產流程。 實驗結果顯示，初代培養中，將田間取回之新梢以0.3% (v/v) Clorox 漂白水滅菌一晚後取5mm 的莖頂為培植體，以MS、2% (w/v) 蔗糖及 4.44 μM BAP為培養基，在25 ℃、光強度 56 μmol m-2 s-1之室內條件下，一個月後的存活率達100%，此時芽長0.9公分，具3葉。將單芽切下以MS、3% (w/v) 蔗糖及 4.44 μM BAP為培養基進行繼代增殖，30天後可長出4個長2.1公分，具5.3葉的芽。上述增殖的枝條於含有MS培養基、3% (w/v) 蔗糖、無植物生長調節劑下，30天後瓶內發根率可90%，平均根長3.3-5.7公分，根數約4.8-6.8；瓶內發根苗出瓶，種植於混合蛭石：珍珠岩：泥炭土（體積比為1:1:1）的濕潤介質並於辦公室（27℃）內健化，30天後生活率可達94%以上。其瓶外發根以取3-4葉片的瓶內增殖枝條培植體、沾2000 ppm IBA之滑石粉，經扦插於同上介質，於辦公室（27℃）健化30天後，發根率可達100%，平均根長2.2公分，根數約8.3根。不論瓶內或瓶外的已發根苗，再移至溫室內進行第二階段健化，30天後之成活率均達100%，隨後將之定植於田間，最終成活率亦高達92%。本研究已首次成功建立’枝垂桑’自莖頂培養、繼代增殖、發根、馴化乃至田間定植之高效能微體繁殖體系。Table of Contents i List of Tables iii List of Figures v Abstract vi 摘要 viii Introduction 1 Literature review 6 Chapter I Sterilization and initial culture 17 Abstract 17 Introduction 18 Materials and Methods 20 Experiment 1. Influence of sterilization method on the explants and the effect of different concentrations of BAP on initial cultures of M. alba var. Shidareguwa 20 Experiment 2. Effects of shoot growth on initial culture duration in vitro 21 Results 22 Discussion 31 Chapter II Shoot multiplication 33 Abstract 33 Introduction 34 Materials and Methods 36 Experiment 1. Effects of MS and sucrose level on shoot multiplication 36 Experiment 2. Effects of TDZ concentrations on shoot multiplication 36 Experiment 3. Effects of BAP concentrations on shoot multiplication 37 Experiment 4. Effects of coconut water on shoot multiplication 37 Experiment 5. Multiple rates in subculture times of ''Shidareguwa'' mulberry 37 Results 39 Discussion 56 Chapter III Root induction and acclimation 58 Abstract 58 Introduction 60 Materials and Methods 62 Experiment 1. Effects of different concentrations of IBA, NAA and 2,4-D on root induction in vitro 62 Experiment 2. Effects of MS and NAA on root induction in vitro 62 Experiment 3. Effects of activated charcoal carbon on root induction in vitro 63 Experiment 4. Effects of explant types and IBA concentration on root induction ex vitro 63 Experiment 5. Acclimation of plantlets 64 Results 65 Discussion 78 Chapter VI General Conclusions and Further Studies 82 References 85 Appendix 9