Volume 5 Special Issue 1 2011
Focus on Tree micropropagation and tissue culture

ISBN 978-4-903313-64-1

How to reference: Vijayan K, Tikader A, Teixeira da Silva JA (2011) Application of Tissue Culture Techniques for Propagation and Crop Improvement in Mulberry (Morus spp.). In: Nageswara-Rao M, Soneji JR (Eds) Focus on Tree Micropropagation and Tissue Culture. Tree and Forestry Science and Biotechnology 5 (Special Issue 1), 1-13

Guest Editors

Madhugiri Nageswara-Rao, Jaya R. Soneji

University of Florida, IFAS, Citrus Research & Education Center, USA

www.crec.ifas.ufl.edu/

CONTENTS AND ABSTRACTS

Kunjupillai Vijayan, Amalendu Tikader (India), Jaime A. Teixeira da Silva (Japan) Application of Tissue Culture Techniques for Propagation and Crop Improvement in Mulberry (Morus spp.) (pp 1-13)

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Invited Review: Mulberry (Morus spp.) is an economically important tree grown widely in China, India and several other countries in Asia. The major economic product of mulberry is its leaf which is used for feeding the monophagous silkworm Bombyx mori L. Mulberry leaf is also used as fodder for livestock. Mulberry fruit is good for human consumption. Although mulberry is amenable to both sexual and asexual methods of reproduction, due to the high heterozygosity of the parental lines and long juvenile periods, propagation is mainly through stem cuttings or bud grafting. However, success of propagation through stem cuttings is greatly dependent on the genotype, environment, and age of the planting materials. Besides, most of the temperate species are hard to root from the stem cuttings. Micropropagation is seen as a cost effective method for propagation of these species, though a number of factors affect the success. In this article, we summarize these factors along with the causes and remedies for them. Other applications of tissue culture such as germplasm conservation, screening for stress tolerance, triploid developments, genetic transformation, and their impacts on the sericulture industry have also been detailed and discussed.

Ravindra B. Malabadi (India), Jaime A. Teixeira da Silva (Japan) Thin Cell Layers: Application to Forestry Biotechnology (pp 14-18)

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Invited Mini-Review: Thin cell layers or TCLs have been one of the most recent yet important techniques for the tissue culture and micropropagation of select forestry species. In this review, we abridge the current state of application of TCL technology to forest tree biotechnology, focusing on Pinus spp., Paulownia spp. and Populus spp. Where conventional methods have not always produced ideal results in the micropropagation of forestry species, TCL technology has now perfected the ability to control developmental and morphogenetic processes in vitro at a finer scale than the use of conventional explants focusing the size and the origin of the explant to ensure successful regeneration for micropropagation and transformation studies. TCLs are fundamental in implementing lab-based experiments to larger scale field-based applications and its application and proven success with three select forestry and timber species provides a frame-work for the development of the technique for other forestry species.

Inmaculada Hernández, Beatriz Cuenca, Elena Carneros, Nieves Alonso-Blázquez, Mar Ruiz, Cristina Celestino, Luis Ocaña, Jesús Alegre, Mariano Toribio (Spain) Application of Plant Regeneration of Selected Cork Oak Trees by Somatic Embryogenesis to Implement Multivarietal Forestry for Cork Production (pp 19-26)

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Invited Review: To implement multivarietal forestry, the availability of an efficient and economically viable methodology for vegetative propagation is required. In addition field tested varieties have to be developed. Somatic embryogenesis is increasingly being considered as the most appropriate way of cloning forest species. The cork oak (Quercus suber L.), one of the most important Mediterranean tree species, has been considered recalcitrant to vegetative propagation for long time. In this article we review the current status of plant regeneration by somatic embryogenesis in this species, and report on a joined effort between IMIDRA, a public research institution, and TRAGSA, a public company, to apply this tissue culture derived methodology to cork oak genetic improvement. At IMIDRA, a protocol to induce somatic embryogenesis in leaves of adult cork oak trees, applicable to any genotype, that allow to clone elite trees has been developed. In the frame of the project SEFEAL-2 led by TRAGSA, this protocol is being implemented with the goal of cloning trees, selected for their cork quality and productivity in Extremadura (Spain). Data on current frequencies of induction of embryogenic lines, and of plant conversion and survival at the operational level are provided.

Ravindra B. Malabadi (India), Jaime A. Teixeira da Silva (Japan), Gangadhar S. Mulgund (India) Induction of Somatic Embryogenesis in Pinus caribaea (pp 27-32)

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Original Research Paper: Brassinosteroids are of ubiquitous occurrence in plants and elicit a wide spectrum of physiological responses including somatic embryogenesis in conifers. This study highlights the successful brassinolide-mediated stimulation of embryogenesis in all the five genotypes of Pinus caribaea tested. 24-epiBrassinolide at 2.0 µM with 9.0 µM 2, 4-dichlorophenoxy acetic acid enhanced the formation of embryogenic tissue from mature zygotic embryos on half-strength MSG basal medium. However, the frequency of somatic embryogenesis was not similar in all the five genotypes tested. The highest percentage of somatic embryogenesis (87.0 ± 1.8) was recorded in PC 05 genotype. On the other hand the lowest percentage of somatic embryogenesis (70.0 ± 1.7) was obtained in PC11 genotype. The developed somatic embryos on maturation medium after 12 to 14 weeks in all the five genotypes tested. Therefore, 24-epiBrassinolide can be used as growth regulator in conifer somatic embryogenesis for improving the initiation of embryogenic cultures of recalcitrant pines for the commercial forestry programmes particularly in Indian subcontinent.

C. Anjaneyulu, Charu C. Giri (India) Direct Somatic Embryogenesis from Mature Zygotic Embryo and Conversion to Plants in Medicinal Tree Terminalia chebula Retz. (pp 33-37)

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Original Research Paper: Terminalia chebula Retz. mature zygotic embryo (MZE) explants were evaluated for induction of direct somatic embryogenesis on Murashige and Skoog medium (MS) supplemented with 3% (w/v) sucrose, various combinations and concentrations of 2,4 dichlorophenoxy acetic acid (2,4-D) and kinetin (Kn). Seeds of T. chebula collected from different geographic and climatic zones such as Srisailam Tiger Forest Reserve, Srisailam, Andhra Pradesh, forest area in eastern part of Mayurbhanj district of Orissa and Ananthagiri Hill forest area, Vikarabad, Andhra Pradesh, India. Seeds of geographic zone in Mayurbhanj district of Orissa showed maximum frequency of direct somatic embryo induction (62.00 ± 1.15 fully developed somatic embryos per explants) and was obtained on MS medium containing 1.0 mg/l 2,4-D and 0.01 mg/l Kn after 6 weeks of culture. The influence of seed age (duration after collection) as MZE source on direct somatic embryogenesis revealed that one-month old MZEs promoted maximum frequency; however, this declined with an increase in age. Somatic embryo maturation was obtained on MS media supplemented with 50 g/l sucrose. Direct and indirect (via intervening callus phase) somatic embryogenesis was a parallel process. A maximum of 26.33 ± 1.20 explants showed direct somatic embryogenesis compared to 80.0 ± 2.33 indirect somatic embryogenesis. Direct somatic embryogenesis can be advantageous in terms of curtailing more than four weeks of culture passage in vitro compared to indirect somatic embryogenesis. Conversion of somatic embryos to plants was possible on ½-strength MS medium fortified with 0.5 mg/l BA (6-benzyladenine) and 30 g/l (w/v) sucrose. Direct somatic embryogenesis and the conversion of somatic embryos to plants is reported for the first time in T. chebula.

B. Shyamkumar, Charu C. Giri (India) High Frequency Shoot Proliferation, Rooting, Acclimatization and Field Establishment of Terminalia chebula: a Tree of Pharmaceutical Importance (pp 38-44)

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Original Research Paper: High frequency axillary shoot proliferation from cotyledonary nodes was achieved in Terminalia chebula Retz. Repeated subculture of cotyledonary node explants to fresh media resulted in formation of enormous new shoots. About 44.33 ± 0.88 first generation shoots could be obtained from a single cotyledonary node explants after 12-14 weeks of culture. Highest rooting percentage of 93.4 ± 0.9, 75.3 ± 0.88 in in vitro propagated shoots was obtained on media containing mannitol 1.0% (w/v) and 0.3% (w/v) polyvinyl pyrrolidone (PVP), respectively. Acclimatized plants showed 98.4% survival rate in glasshouse conditions. Histological/anatomical studies clearly indicated the multiple shoot induction pathway regeneration with out intervening callus phase from cotyledonary node explants. Plants hardened in the poly-bags containing soil and vermi-compost in the portable mist chamber showed 84.4% survival rate in field conditions. Nearly 45% of the plants were finally established in the field conditions at CPMB, Osmania University, Hyderabad, India. Plants were also transferred to different climatic locations and established at SAIRD, Gaddipally, Nalgonda District, A.P., India. The field established plants showed early flowering and fruit setting. The present study describes a rapid method for generating planting stock material for forestry and their subsequent establishment in field conditions.

Fatima Shirin, Nitish Parihar, P. K. Rana, S. A. Ansari (India) In Vitro Shoot Regeneration from Embryonic Axis of a Multipurpose Vulnerable Leguminous Tree, Saraca indica L. (pp 45-48)

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Original Research Paper: Saraca indica L. syn. Saraca asoca (Roxb.) De Wilde (Caesalpinaceae) is a commercially important evergreen medicinal tree. The annual demand for its bark is 10,724 tonnes and is growing at 15% per annum in India. Commercial exploitation for its bark has led to it becoming vulnerable, resulting in poor regeneration in its natural habitat. Tissue culture offers a viable alternative for production of a large number of plantlets within a short period of time. The present paper describes the formation of shoots through embryonic axis under the influence of two cytokinins, viz., 6-benzyladenine (BA) and zeatin (Zn). Embryonic axes were excised from one-month old immature seeds and a 1-2 mm cut was introduced at the radicle end. The embryonic axis was inoculated onto B₅ medium supplemented with BA (0 and 2.5 µM) and Zn (0, 5, 10 and 20 µM) to study the effect on direct or indirect organogenesis. BA had a significant effect on the number of shoots formed and callus formation (%). After one month B₅ medium supplemented with 2.5 µM BA regenerated both shoots and callus from embryonic axes. The dose of Zn and its interaction with BA did not significantly affect callus formation, shoot formation or the number of shoots. These shoots were multiplied further on 2.5 µM BA supplemented medium.

Rajesh Yarra, Mahender Aileni, Venugopal Rao Kokkirala, Pavan Umate, Anil Kumar Vemunoori, Sadanandam Abbagani (India) Micropropagation of Cochlospermum religiosum (L.) Alston (pp 49-52)

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Original Research Paper: An in vitro micropropagation system has been developed for Cochlospermum religiosum (L.) Alston using node segments, obtained from 15-day-old aseptically grown seedlings, cultured on MS medium containing 6-benzyladenine (BA) and kinetin (Kn) either alone, or in combination. The regeneration medium with combined BA (4.44 µM) and Kn (4.65 µM) showed highest mean number of shoots (3.01 ± 0.32), increased shoot length (1.21 ± 0.06), and 80% regeneration response within 21 days. To initiate subculture, when separated and transferred to fresh medium with different levels of BA (4.44, 6.66 and 8.87 µM) and Kn (4.65, 6.97 and 9.29 µM), the medium containing BA (8.87 µM) and Kn (4.65 µM) resulted in further elongation of regenerated shoots. This phytohormone combination caused an increase in shoot length (2.23 ± 0.05) within 42 days from the beginning of culture. The regenerated shoots showed optimal rooting response (80%) on medium containing half strength MS salts, 0.8% agar and 4.90 µM indole-3-butyric acid (IBA). The plantlets established in vitro were transferred to pots containing sterilized soil and vermiculite (1:1) mixture. On transfer to the greenhouse, 65-70% plants survived. The present investigation for the first time describes an efficient micropropagation protocol for C. religiosum.

Kamlesh Kanwar, Anue Parmar, Sunita Chandel, Raj Deepika, S. V. Bhardwaj (India), Jaime A. Teixeira da Silva (Japan), Vipasha Verma (India) In Vitro Selection for Resistance against Fusarium equisetii in Robinia pseudoacacia L. (pp 53-57)

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Original Research Paper: The objective of this study was to develop resistance in vitro against Fusarium equisetii in Robinia pseudoacacia L. Callus was induced from cotyledon explants of R. pseudoacacia, a leguminous landscaping tree, on solid MS medium supplemented with different concentrations and combinations of 1-naphthalene acetic acid (NAA; 0.1-0.5 mg/l) and N⁶-benzyladenine (BA; 0.1-0.3 mg/l). Among them, 1.0 mg/l NAA and 1.0 mg/l BA was the best combination for callus induction, which was friable and light brown. Callus fresh weight increased and turned pale yellow after subculture. The effect of different concentrations of a toxic culture filtrate of Fusarium equisetii (Corda) Sacc. was investigated by plating friable callus on selective medium. Resistant calli were selected at 10% of the culture filtrate on which 15.33% calli survived, although no callus formed at higher concentrations of toxic culture filtrate. Shoots, which regenerated from resistant calli on Murashige and Skoog (MS) medium supplemented with 0.05 mg/l NAA and 0.6 mg/l BA, could root on half-strength MS medium containing 0.02% activated charcoal. Selected resistant plantlets, which survived on 2.5-10% of the culture filtrate compared to the control, showed reduced wilt incidence (approximately 70%) indicating a resistant response in tissues towards F. equisetii.

Vinod Singh Gour (Tanzania/India), Tarun Kant (India) Efficacy of Low Cost Gelling Agents and Carbon Source Alternatives during in Vitro Rooting of Balanites aegyptiaca and Phyllanthus emblica Microshoots (pp 58-60)

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Short Communication: Several attempts have been made to reduce the cost of tissue culture raised plants. The efforts have largely concentrated around using low cost alternatives to agar (gelling agent) and sucrose (carbon source). Isabgol-husk, guar gum and sago powder were used as low cost gelling alternatives and table sugar, raw sugar and jaggary were tested as low cost carbon sources in tissue culture media. These low cost alternatives were tested during in vitro rooting of microshoots of two plants of arid and semi-arid regions of India: Balanites aegyptiaca and Phyllanthus emblica. Sago powder, guar gum and isabgol husk solidified the media properly at 15.0, 5.0 and 5.0% (w/v), respectively. In the presence of isabgol, rooting response in microshoots of B. aegyptiaca was equivalent to that of agar and application of isabgol husk can reduce the cost of gelling agents per unit media by ~44%. Further, table sugar can be used for in vitro rooting of both the species at a commercial scale. As far as gelling agents are concerned, isabgol was found to be a suitable gelling agent for rooting with equally good rooting response comparable to agar.

Farida Anwar Meerja, Yuan Song (Canada/China), Susan Sibbald, Jayasankar Subramanian, Lining Tian (Canada) European Plum in Vitro Regeneration from Different Organs (pp 61-64)

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Research Note: Plum (Prunus domestica L) is an important fruit crop in many countries. Several aspects were studied to improve plum in vitro regeneration. Three different media, namely, B5, WPM, and MS, were evaluated for shoot induction from hypocotyls. Use of B5 medium resulted in significantly higher regeneration efficiency compared to other types of media. Thidiazuron (TDZ) was found to be more effective for promotion of shoot induction than 6-benzylaminopurine (BA). Increase of concentration of TDZ did not show an increase in regeneration efficiency. Study was conducted to evaluate shoot induction from epicotyls of stored mature seeds. Shoots were induced from epicotyls of all 13 plum varieties evaluated. However, strong genotype difference exits for shoot development via epicotyls. Full plants were recovered from the shoots induced from epicotyls.

Regular papers

Adele Muscolo, Carmelo Mallamaci, Maria Sidari, Roberto Mercurio (Italy) Effects of Gap Size and Soil Chemical Properties on the Natural Regeneration in Black Pine (Pinus nigra Arn.) Stands (pp 65-71)

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Original Research Paper: This paper deals with the problem of black pine (Pinus nigra Arn.) stand re-naturalization in the Apennines of Central Italy. Gap cuttings of two different sizes (gap diameter (D) to stand height (H) ratios were 0.75 and 1.0) in age class stands (50 and 90 years old) have been carried out, studying the soil properties and the tree regeneration dynamics in the different gaps 7 years after the beginning of the experiment. The small gaps D:H = 0.75 had the greater concentrations of total phenols, polyphenols and fulvic acids. In contrast, a greater amount of humic acids was detected in the medium gaps D:H = 1. Results indicate that black pine was the best regenerated species in gaps with D:H = 1 and its presence was increasing. Some broadleaves, pubescent oak (Quercus pubescens) and holm oak (Quercus ilex) particularly and hop-hornbeam (Ostrya carpinifolia) and manna-ash (Fraxinus ornus) in a subordinate way, were also present in the gaps. These results confirm that the gap cutting system represents an effective tool for the natural regeneration priming and a treatment with low environmental impact which provides suitable condition for the re-naturalization of the conifer monocultures.

Felix D. Ugese, Paul K. Baiyeri, Benjamin N. Mbah (Nigeria) Variability in Seedling Growth of Seeds of Shea Butter Tree (Vitellaria paradoxa C. F. Gartn.) Sourced from Nine Locations in Nigeria (pp 72-77)

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Original Research Paper: Provenance evaluation is essential in identifying genotypes with superior seedling growth. The seedling performance of nine accessions of Vitellaria paradoxa obtained from Southern Guinea Savanna, Northern Guinea Savanna and the Sudan Savanna of Nigeria were investigated at two contrasting locations – Makurdi and Nsukka – between July 2006 and July 2007. Seedling emergence of the Makurdi trial was monitored while seedling growth at both sites was monitored at monthly intervals from the 6^th to 12^th month after sowing. Results of analysis of variance showed wide variation in emergence parameters with the Akwanga, Makurdi, Jalingo and Yola seed sources giving significantly higher emergence percentage (E %). Most accessions had emergence index (EI) values of 100 or more with corresponding high emergence rate index (ERI) scores. The main effect of location on seedling growth showed the superior performance of seedlings grown at Makurdi over those grown at Nsukka. There was a significant interaction between location and accession. Generally, while the Akwanga, Kachia and Yola provenances gave better seedling vigour at Makurdi, at Nsukka, it was Akwanga, Kachia and Jalingo suggesting the existence of genotype x environment interaction.

Ahmad Dadashpour, Alireza Talaei, Mohammad Ali Asgari-Sarcheshmeh, Ali Shahi-Gharahlar (Iran) Evaluation of Agropomological Properties of Some Commercial Apple Cultivars in an Intensive Planting System in Karaj Climate (pp 78-81)

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Original Research Paper: Nowadays apple growers tend to use intensive planting systems and dwarfing rootstocks in order to increase yield and decrease production costs. A V-shape system represents an efficient and popular option to increase yield and fruit quality. Hence, this paper attempts to compare some vegetative, yield and fruit properties of five apple cultivars (‘Golab-kohans’, ‘Fuji’, ‘Gala’, ‘Starking’ and ‘Delbar estival’) grown at a horticultural research station in Karaj, Iran. The experimental apple cultivars were grafted onto an M.9 rootstock trained in a V system. All trees were planted in the winter of 2005 using drip irrigation. ‘Golab-kohans’ formed the highest trees (278.63 cm), trunk cross sectional area (7.31 cm²), mean shoot length (100.58 cm) and pH (4.85). Also ‘Delbar estival’ had the highest yield (0.98 kg), yield efficiency (0.55 kg/cm²), fruit weight (131.30 g), fruit length (5.91 cm), fruit diameter (6.72 cm) and L/D (0.87). In addition, ‘Fuji’ had the highest dry matter (21.71%) and fruit sunburn (56.92%). ‘Golab-kohans’ had the highest ash (0.66) and TSS (16.12) levels. ‘Starking’ had the highest fruit firmness (13.60 kg cm^-2) and titrable acid (0.73). Consistently, results revealed that among the investigated cultivars, ‘Delbar estival’ can be introduced as a productive cultivar for the V system in Karaj’s climatic condition.

Appaji Nanda, Hiregouja M. Prakasha, Yelugere L. Krishna Murthy (India) Leafing Patterns and Seasonality at Community Level in a Tropical Dry Deciduous Forests of Bhadra Wildlife Sanctuary, Karnataka, Southern India (pp 82-87)

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Research Note: This study describes the role of mean temperature and total rainfall patterns on the distribution and duration of various leafing phenophases of individual trees recorded at the community level in a tropical dry deciduous forest of Bhadra Wildlife Sanctuary, southern India. Plant stress to surrounding environment was assessed by individual trees’ phenological response. Regression analysis was performed to examine how variations in rainfall and temperature influenced leafing phenology. We also investigated seasonality of various phenophases to know the cyclicity and strength of seasonality: Leaf initiation and leaf expansion in the beginning and late April, leaf senescence in the middle of January and leaflessness in March. Seasonality study indicates how tropical dry deciduous forest trees respond to their surrounding environment: a strong seasonality in leafless event followed by leaf senescence, leaf initiation and expansion were observed. Overall our study suggests that the response/ sensitivity of tropical dry deciduous forest foliar phenophases is an indication of regional environmental changes.

Rosario Nicoletti (Italy) Endophytic Occurrence of Sphaceloma murrayae on White Willow (Salix alba) in Italy (pp 88-89)

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Research Note: Despite being considered a common disease in countries such as New Zealand, Argentina and the United States, the occurrence in Europe of grey scab of willows incited by Sphaceloma murrayae has been reported quite recently, and to date no observation concerns Italy. An endophytic isolate of this foliar pathogen was recovered from the secondary branch of a white willow tree (Salix alba) in Basilicata. This finding represents the first documented report of S. murrayae from Italy and of its endophytic occurrence in S. alba.