International Journal of
Biotechnology and Molecular Biology Research

  • Abbreviation: Int. J. Biotechnol. Mol. Biol. Res.
  • Language: English
  • ISSN: 2141-2154
  • DOI: 10.5897/IJBMBR
  • Start Year: 2010
  • Published Articles: 100

Full Length Research Paper

Induction and regeneration of somatic embryos from Vitex doniana (Lamiaceae) leaf explants

Colombe Dadjo
  • Colombe Dadjo
  • Institute of Basic Sciences, Technology and Innovation, Pan African University, P. O. Box 62000-00200, Nairobi, Kenya.
  • Google Scholar
Jane Kahia*
  • Jane Kahia*
  • World Agroforestry Centre (ICRAF), Cote d? Ivoire Country Program Cocody Mermoz, Abidjan, Cote d?Ivoire.
  • Google Scholar
Catherine Muthuri
  • Catherine Muthuri
  • ICRAF World Agroforestry Centre (ICRAF), P. O. Box 30677-00100, Nairobi, Kenya.
  • Google Scholar
Lucien Diby
  • Lucien Diby
  • World Agroforestry Centre (ICRAF), Cote d? Ivoire Country Program Cocody Mermoz, Abidjan, Cote d?Ivoire.
  • Google Scholar
Christophe Kouame
  • Christophe Kouame
  • World Agroforestry Centre (ICRAF), Cote d? Ivoire Country Program Cocody Mermoz, Abidjan, Cote d?Ivoire.
  • Google Scholar
Peter Njenga
  • Peter Njenga
  • Jomo Kenyatta University of Agriculture and Technology, P. O. Box 62000-00200, Nairobi, Kenya.
  • Google Scholar
Modeste Kouassi
  • Modeste Kouassi
  • Centre National de Recherche Agronomique (CNRA), Laboratoire Central de Biotechnologies (LCB), 01 BP 1740 Abidjan 01, Cote d?Ivoire.
  • Google Scholar


  •  Received: 20 October 2014
  •  Accepted: 01 May 2015
  •  Published: 31 May 2015

 ABSTRACT

The present study was conducted with the aim of evaluating some of the factors that influence induction and regeneration of somatic embryos in Vitex doniana since there are no available reports on tissue culture of this tree species. Leaves from plants growing under temporary shed were cultured on Murashige and Skoog supplemented with silver nitrate and four amino acids (proline, tryptophan, lysine and leucine) at varying concentrations, 0.11 mg/l thidiazuron, 2% sucrose and 100 mg/l myo-inositol in separate experiments. The explants cultured on media supplemented with tryptophan at 30.6 mg/l produced the optimal (6.5) number of embryos per explant. This number was fivefold more than the number obtained in the control. On the other hand, it was observed that the explants on media supplemented with silver nitrate at 8.45 mg/l gave the same mean (6.5) number of embryos per explant. These first ever results on the induction of somatic embryo in V. doniana could be used for mass propagation and to select useful traits of this tree species at the cellular level. However, further work needs to be done on the conversion of the regenerated embryos.

 

Key words: Amino acids, leaf explant, silver nitrate, somatic embryogenesis, Vitex doniana.


 INTRODUCTION

Many neglected and underutilized wild species (NUS) are nutritionally rich (Ghane et al., 2010; Johns and Eyzaguirre, 2006). Therefore, their erosion can have immediate consequences on the nutritional status and food security of the poor and their enhanced use can bring about better nutrition and fight hunger. Even though the link between agrobiodiversity and diet diversity is not automatic (Burchi et al., 2011), it is agreeable that the diminution of agrobiodiversity, to some extent, places considerable strain on the ease with which households are able to enjoy diversified, balanced diets. Awareness of the importance and value of crop wild relatives and of the need to conserve them has increased one of such high value plant that is in dire need for conservation, Vitex doniana Sweet (Black plum). It had been considered to belong to Verbenaceae family by different authors but in recent works, it has been transferred into Lamiaceae based on different evidences (Wagstaff et al., 1998). It is the most abundant and widespread Vitex species in Africa (Orwa et al., 2009). Black plum (V. doniana) is an indigenous species important for the livelihoods of rural populations in Benin Republic in particular and West Africa in general (Codjia et al., 2003). The fruits and leaves are the edible part of the trees. They are either eaten raw or after processing. The plant is also widely used in traditional system for medicinal purposes. The leaves, fruits, roots, barks and seed of the plant have been used as medication for liver disease, anodyne, stiffness, leprosy, backache, hemiplegia, conjunctivity, rash, measles, rachitis, febrifuge, as tonic galactagogue to aid milk production in lactating mothers, sedative, digestive regulator, treatment of eye troubles and kidney troubles. It has also been used for treatment of conditions such as infertility, anemia, jaundice, dysentery, gonorrhea, headaches, diabetes, chickenpox, rash and fever (Louppe et al., 2008; Orwa et al., 2009). Despite the widely known nutritional, medicinal and economic uses of V. doniana products, the species is still under-utilized, unimproved. To date, the species has been chosen as a model species to be domesticated in Benin (Codjia et al., 2003; Dadjo et al., 2012).The strong anthropic pressure affecting this species has caused its numbers to fall increasingly in its natural environment (Achigan-Dako et al., 2010). The planting of seedlings is negligible and the seeds of this tree have a very weak germinating capacity (Thies 1995). Sanoussi et al. (2012) reported that macropropagation rate of this tree species by stem cuttings is slow. Therefore, there is need to seek for alternative propagation methods. Tissue culture methods are considered as the most promising means to protect and propagate tree species of economic interest. In vitro propagation through somatic embryogenesis is the most feasible alternative to the other in vitro methods (Zimmerman, 1993, Saiprasad, 2001). Somatic embryos are widely considered to be of single cell origin; hence this is advantageous for transformation studies. Moreover, the process of somatic embryogenesis offers a mean to propagate large numbers of transgenic plants over a short period of time.

The success in tissue culture depends on the effectiveness of the sterilization methods used on the explants prior to culture initiation (Yildiz and Celal, 2002). Sterilization is the process of eliminating contamination from explants before establishment of in vitro cultures. Various sterilization agents such as calcium hypochlorite and sodium hypochlorite are commonly used to decontaminate the tissues.

Ethylene is known to reduce somatic embryogenic competence in many plants and the use of silver nitrate (AgNO3), an ethylene action inhibitor (Beyer, 1976), has been shown to influence in vitro somatic embryogenesis (Kong et al., 2012). It has also been reported that amino acids play a key role in plant growth and development because they are good source of nitrogen (Kirby et al., 1987; Shanjani, 2003). Amino acids such as glutamine, proline and tryptophan, have been identified as enhancers of somatic embryogenesis in some species (Deo et al., 2010). Their efficacy in embryogenesis has been attributed to their contribution to various cellular processes such as improving cell signaling processes in various signal transduction pathway (Lakshmanan and Taji, 2000) and as precursor molecules for certain growth regulators. So far, there are no reports on tissue culture of V. doniana. During the current study, we hypothesized that the various concentrations of AgNO3 and some amino acids could enhance induction and regeneration of somatic embryos in V. doniana. Therefore, the aim of this study was to evaluate the effects of various concentrations of AgNO3 and some amino acids on induction and regeneration of somatic embryos in V. doniana


 MATERIALS AND METHODS

V. doniana seedlings (2-4 years old) originally wildings collected from the field in Glo in the south part and Cove in the central part of Benin were transported to Abidjan, Côte d’ Ivoire where they were maintained in a temporary shed and watered daily for one month before harvesting the leaves.

 

Explant preparation and surface sterilization

Healthy looking young leaves (2nd pair) were collected and cleaned with cotton wool soaked with liquid soap. Thereafter, they were immersed in 0.5% (w/v) fungicide (Ridomil) containing two drops of Tween-20 (wetting agent) for an hour. The leaf explants were then transferred to the lamina flow cabinet for surface sterilization. Pre sterilization was carried using 70% ethanol solution for thirty seconds and then rinsing twice with sterile distilled water. The explants were subjected to further sterilization using varying (1, 1.5 and 2%) concentrations of calcium hypochlorite (CaOCl2) containing 2-3 drops of Tween 20 and varying time duration. In an attempt to increase the number of clean explants, double sterilization (two steps) was conducted. This involved sterilizing the explants using 2% calcium hypochlorite for 30 min rinsing twice with sterile distilled water followed by quick dip in 70% ethanol. They were then subjected to a second step, by sterilizing them with 2% calcium hypochlorite for 15 min and finally rinsing with sterile distilled water four times.

 

Media preparation and culture conditions

Silver nitrate (at concentrations 8.45; 16.9 and 25.35 mg/l) and four amino acids namely proline (5.75; 11.5 and 17.25 mg/l), tryptophan (10.2; 20.4 and 30.6 mg/l), lysine (7.3; 14.6 and 21.9 mg/l) and leucine (6.55; 13.1 and 19.65 mg/l) were added to half strength Murashige and Skoog (MS) (1962) medium supplemented with 100 mg/l myo-inositol, 0.11 mg/l TDZ and 2% sucrose in separate experiments. The pH of the medium was adjusted between 5.7 and 5.8 prior to the addition of the solidifying agent, and autoclaved at 121°C for 15 min. The MS medium without AgNO3 and amino acids is referred to as control. All cultures were incubated in a dark room maintained at 25 ± 2°C.

 

Experimental design, data collection and analysis

Completely randomized design was used for all the experiments. Each single explant was considered as experimental unit. Twenty (20) replicates per treatment were used at the outset of experiments and each treatment repeated at least twice. The data were subjected to one-way analysis of variance (ANOVA) and the significant differences between treatment means were assessed by using Minitab version 14 Software. The results of the sterilization experiment are expressed as percentage (%) while for regeneration of somatic embryos data are presented as means ± standard error (SE). 


 RESULTS

Surface sterilization

The highest  percentage  (70%)  of  clean  leaf   explants were obtained when 2% calcium hypochlorite was used for 20, 25 and 30 min, respectively (Table 1). However, this percentage decreased further and after two weeks only 50% clean explants were observed. The evaluation of double sterilization treatment resulted in increase in percent clean explants from 50 to 91% after one month and this procedure was used for all the subsequent experiments.

 

 

Effect of AgNO3 on induction and regeneration of somatic embryos

The addition of AgNO3 to the culture medium had a significant effect on the mean number of embryos per explant (Table 2). The percent embryogenic cultures decreased with increasing AgNO3 concentration. The highest (91 and 90 %) frequency of embryogenic cultures were observed in the control and in the media supplemented with 8.45 mg/l AgNO3. The later AgNO3 concentration produced the highest (6.5) mean number of embryos. On the other hand, the lowest (1.2) mean number of somatic embryos was produced in the control. Significant differences were detected between the number of somatic embryos per explant at 8.45 mg/l AgNO3 and the other concentrations of AgNO3 at p < 0.01. Callus was observed from the cut edges of the leaf and globular embryos were obtained four weeks after culture (Figure 1A and B). Cotyledonary stage of embryos was observed two months after culture as shown in Figure 1C.

 

 

 

Effect of some amino acids induction and regeneration of somatic embryos

Among the four amino acids evaluated, the highest leucine concentration (19.65 mg/l) was found to inhibit embryo formation (Table 3). However, when leaf explants were cultured on media supplemented with the lower concentrations (6.55 and 13.1mg/l), the mean number of embryos obtained were double those obtained in the control. It was observed that increasing the concentration of proline from 5.75 to 17.25 mg/l decreased significantly the mean number of embryos. Leaf explants cultured on medium supplemented with 30.6 mg/l tryptophan produced the highest (6.5) mean number of somatic embryos which was fivefold more than the embryos obtained in the control.

 

 


 DISCUSSION

The current study was conducted with the aim of evaluating some of the factors that influence induction and regeneration of somatic embryos since there were no available reports on tissue culture of V. doniana. Before any explants is placed into culture, it is essential to destroy all microorganisms and the success in tissue culture depends on the effectiveness of the sterilization methods used on the explants prior to culture initiation (Yildiz and Celal, 2002). The outer surface of plants growing under natural or greenhouse conditions is normally infected with spores and other microbial cells. The use of field grown plants as a direct source of explant material for obtaining ‘clean’ explant, presents a major challenge. Previous attempts to initiate clean explants from field grown coffee, especially those from canopy close to the ground, resulted in 100% contamination (Kahia, 1999). In an attempt to obtain clean in vitro cultures, sources of contamination other than surface contaminants need to be considered. Even if the surface of the explant is effectively sterilized, the contaminants could emanate from the inner tissues when the plant materials are dissected into small explants after the surface sterilization. Systemic contaminants, for example, are not eliminated by surface sterilization (Webster et al., 2003). For this reason, a systemic fungicide such as Ridomil used in this study can be helpful to control the incidence of internal fungal infection in explants. The disinfectant widely used for surface sterilization of explants in tissue culture is sodium hypochlorite which dates back to the mid-18th century (Miche and Balandreau, 2001). It is usually purchased as household laundry bleach and as such it is readily available and can be diluted to proper concentrations. A balance between concentration and time must be determined empirically for each type of explant because of phytotoxicity. Previous reports indicate that bleach (1.4% NaOCl solution for 1 min) was found to be effective in sterilizing greenhouse-derived leaf explants in Aquilaria crasna and Aquilaria sinensis  (Okudera and Ito, 2009). On the other hand, effective sterilization was achieved by using 50% bleach for 20 min on shoot tips of Aquilaria hirta, from greenhouse (Hassan et al., 2011) while leaf explants of Allanblackia stuhlmannii were best surface sterilized using 8% sodium hypochlorite (Neondo et al., 2011). The other sterilant used for decontaminating explants is calcium hypochlorite. It is known to be less injurious to plant tissues and is generally used at a concentration of 3.25% (CSS 451 2009). During the present study, calcium hypochlorite was used as attempts to use the commercial bleach even at very low concentrations was found to be phytotoxic and led to the death of explants. The lower concentrations of calcium hypochlorite were not effective in decontaminating the explants. These results concur with those of Mihaljevi? et al. (2013) who reported high percent sterilization (80%) of sour cherry nodes when a high (3%) concentration of CaOCl2 was used. A two-step sterilization was adopted in the work being reported in order to increase the percent clean explants and using this procedure, 91% clean explants was recorded. Similar results were reported by Nieves and Evalour (2011) who recorded 90% clean explants from cotyledon of Moringa oleifera using two steps comprising of 5% Ca(OCl)2.

There are conflicting reports on the use of silver nitrate in induction and regeneration of somatic embryos. When added at low concentrations of 5 to 50 µM (0.845 to 8.45 mg/l), it was found to inhibit somatic embryo formation in Coffea canephora leaf explants (Hatanaka et al., 1995). On the other hand, media supplemented with AgNO3 has been shown to improve somatic embryogenesis  in species such as  Triticum durum (Poaceae) (Fernandez et al., 1999), Coffea canephora (Rubiaceae) (Fuentes et al., (2000), Spinacia oleracea (Ishizaki et al., 2000), Carthamus tinctorius (Asteraceae) (Mandal et al., 2001), Paspalum scrobiculatum (Poaceae) (Vikrant, 2002), Bactris gasipaes (Arecaceae) (Steinmacher et al., 2007), Paspalum scrobiculatum (Poaceae) and Eleusine coracana (Poaceae) (Kothari-Chajer et al., 2008), Hedychiurn bousigonianum (Gingeberacea) (Sakhanokho et al., 2009), Gossypium nelsonii (Malvaceae), Gossypium australe (Yan et al., 2010) and Pinus taeda (Pinaceae) (Pullman et al., 2003). Kong and Yeung (1995) reported that 100 µM AgNO3 (16.9 mg/l AgNO3) stimulated embryo formation in white spruce. The exact mechanism by which AgNO3 affects somatic embryogenesis is not completely understood (Kong et al., 2012). During the current study, incorporating 8.45 mg/l silver nitrate in the media led to a fivefold increase in the number of embryos as compared to the control. However, it was observed that increasing the concentration from 8.45 to 25.35 mg/l decreased both the percentage of embryogenic cultures and the mean number of embryos. These results are contrary to those of Kong et al. (2012) who reported that the number of somatic embryos per explant in Manchurian ash increased with increasing AgNO3 concentration. These workers reported that the lowest number of embryos per explant (1.0) was induced at 2.5 mg/l AgNO3 while the higher concentration of 10 mg/l resulted to a threefold increase in the number of somatic embryos with mean (3.86) embryos per explant. Comparable results have also been reported by Ishizaki et al. (2000) in Spinacia oleracea where addition of 10 mM AgNO3 (1.69 g/l AgNO3) to the medium resulted in formation of about three times more embryos as compared to the controls. Fuentes et al. (2000) found that the addition of AgNO3 caused only small modifications in the ionic equilibrium of the medium and concluded the effects of the compound on somatic embryogenesis were not attributable to any substantial changes in available nutrients.

Requirement of exogenous supply of amino acids for in vitro somatic embryogenesis has been reported in a number of plant species (Basu et al., 1989; Claparols et al., 1993). For instance, glutamine was found to be beneficial for embryo development in date palm (El?Shiaty et al., 2004). These workers reported that MS medium supplemented with 100 mg/l glutamine gave the highest (3.33) mean number of embryos. In another report, incorporating proline in sugarcane cultures enhanced somatic embryogenesis (Gill et al., 2004). In Peucedanum oreoselinum, embryo formation and maturation was enhanced by addition of proline in MS medium (Coste et al., 2011). On the other hand, in wheat culture, the efficiency of the amino acids was found to be genotype-based (Duran et al., 2013). Sarker et al. (2007) reported that there were significant responses when L-asparagine at 150 mg/l concentration was used in four popular Bangladeshi wheat cultivars viz Kanchan, Shourav, Gourav and Satabdi. In the work being reported, it was found that supplementing MS media with amino acid promoted the induction and regeneration of somatic embryos. These results concur with those of Shahsavari (2011) who reported that when 100 to 300 µM (20.4 to 61.2 mg/l) tryptophan was added to callus induction medium of rice, there was a great enhancement of the frequency of embryogenic cultures and regeneration of somatic embryos. The lowest concentration of proline evaluated in the current study increased almost three folds the mean number of embryos as compared to the control. However, increasing the proline concentration resulted in decrease in the frequency of embryogenic cultures and the mean number of embryos. These results are contrary to those reported by Chowdhry et al. (2003) who observed that increasing the concentration of proline enhanced embryogenesis  in  rice. Differential  responses of different amino acids indicate the requirement of specific amino acids for somatic embryo regeneration in V. doniana


 CONCLUSION

This study clearly demonstrates that this tree species is amenable to somatic embryogenesis. The results of the current study will be a valuable tool to complement the production of planting materials and thus help in exploiting the medicinal and nutrition value of this tree for the rural poor in Benin and Africa in general. Ex situ and domestication programs of V. doniana could benefit from the findings of the current study. There is however need to do more work on the conversion of somatic embryos.


 CONFLICT OF INTERESTS

The authors hereby declare that there is no conflict of interest.


 ACKNOWLEDGMENTS

The authors thank the World Agroforestry Centre (ICRAF), Côte d’ Ivoire country Program for allowing them to use their facilities to carry out the work. The help rendered by the staff of ICRAF Somatic Embryogenesis Laboratory in Côte d’ Ivoire is highly appreciated.



 REFERENCES

Achigan-Dako EG, Pasquini MW, Assogba-Komlan F, N'danikou S, Yédomonhan H, Dansi A, Ambrose-Oji B (2010). Traditional vegetables in Benin: diversity, distribution, ecology, agronomy, and utilisation. Institut National des Recherches Agricoles du Bénin, Benin.
 
Basu A, Sethi U, Guha-Mukherjee S (1989). Regulation of cell proliferation and morphogenesis by amino acids in Brassica cultures and its correlation with threonine deaminase. Plant Cell Rep. 8:333-335.
Crossref
 
Beyer EM (1976). A potent inhibitor of ethylene action in plants. Plant Physiol. 58:268-271.
Crossref
 
Burchi F, Fanzo J, Frison E (2011). The Role of Food and Nutrition System Approaches in Tackling Hidden Hunger. Int. J. Environ. Res. Public Health 8:358-373. 
Crossref
 
Chowdhry CN, Tyagi AK, Maheshwari N, Maheshwari SC (1993). Effect of L-proline and L-tryptophan on somatic embryogenesis and plantlet regeneration of rice (Oryza sativa L. cv. Pusa 169). Plant Cell Tiss. Org. Cult. 32:357-361.
Crossref
 
Claparols I, Santos M.A, Torne JM (1993). Influence of some exogenous amino acids on the production of maize embryogenic callus and on endogenous amino acid content. Plant Cell Tiss. Org. Cult. 34 (1):1-11.
Crossref
 
Codjia JTC, Assogbadjo AE, Ekue MRM (2003). Diversité et valorisation au niveau local des resource vegetales forestieres alimentaires du Benin. Cahiers Agric. 12:321-331.
 
Coste A, Oltean B, Halmagyi A, Deliu C (2011). Direct somatic embryogenesis and plant regeneration in Peucedanum oreoselinum (L.) Moench. Rom. Biotech. Lett. 16(4):6451-6459.
 

CSS 451 (2009). Biotechnology Applications for Plant Breeding and Genetic. January 13, Course and laboratory orientation; sterile techniques and tissue culture 

View

 
Dadjo C, Assogbadjo AE, Fandohan B, Glèlè Kakaï R, Chakeredza S, Houehanou TD, Van Damme P, Sinsin B (2012). Uses and management of black plum (Vitex doniana Sweet) in Southern Benin. Fruits, 67(4):239-248.
Crossref
 
Deo PC, Tyagi AP, Taylor M, Harding RM, Becker DK (2010). Factors affecting somatic embryogenesis and transformation in modern plant breeding. South Pac. J. Nat. Appl. Sci. 28(1):27-40.
Crossref
 
Duran RE, Coskun Y, Demırcı T (2013). Comparıson of amıno acıds for theır effıcıency on regeneratıon ın wheat embryo culture. Asian J. Plant Sci. Res. 3(1):115-119.
 
El‐Shiaty OH, El‐Sharabasy SF, El‐Kareim AHA (2004). Effect of some amino acids and biotin on callus and proliferation of date palm (Phoenix dactylifera L.) Sewy cultivar. Arabian J. Biotechnol. 7:265-272.
 
Fernandez S, Michaux-Ferriere N, Coumans M (1999). The embryogenic response of immature embryo cultures of durum wheat (Triticum durum Desf): histology and improvement by AgNO3. Plant Growth Regul. 28:147-155.
Crossref
 
Fuentes SRL, Calheiros MBP, Manetti-Filho J, Vieira LGE (2000). The effects of silver nitrate and different carbohydrate sources on somatic embryogenesis in Coffea canephora. Plant Cell Tiss. Org. Cult. 60:5-13.
Crossref
 
Ghane SG, Lokhande VH, Ahire ML, Nikam TD (2010). Indigofera glandulosa Wendl. (Barbada) a potential source of nutritious food: underutilized and neglected legume in India. Genet. Resour. Crop Evol. 57(1):147-153.
Crossref
 
Gill NK, Gill R, Gosal SS (2004). Factors enhancing somatic embryogenesis and plant regeneration in surgarcane (Saccharum officinarum L.). Indian J. Biotechnol. 3:119-123.
 

Hassan NH, Ali NAM, Zainudin F, Ismail H (2011). Effect of 6-benzylaminopurine (BAP) in different basal media on shoot multiplication of Aquilaria hirta and detection of essential oils in the in vitro shoots. Afr. J. Biotechnol. 10(51):10500-10503.

View

 
Hatanaka T, Sawabe E, Azuma T, Uchida N, Yasuda T (1995). The role of ethylene in somatic embryogenesis from leaf discs of Coffea canephora. Plant Sci. 107:199-204.
Crossref
 
Ishizaki T, Komai F, Msuda K (2000). Exogenous Ethylene Enhances Formation of Embryogenic Callus and Inhibits Embryogenesis in Cultures of Explants of Spinach roots. J. Am. Soc. Hort. Sci. 125(1):21-24.
 
Johns T, Eyzaguirre PB (2006). Symposium on Wild-gathered plants: basic nutrition, health and survival" Linking biodiversity, diet and health in policy and practice. Proceed. Nutr. Soc. 65: 182-189.
Crossref
 
Kahia WJ (1999). In vitro propagation of the new Coffea arabica cultivar- Ruiru 11. PhD thesis. University of London.
 
Kirby EG, Leustek T, Lee MS (1987). Nitrogen nutrition. In: Cell and Tissue Culture in Forestry. Volume 1. Edited by Bonga JM, DJ Durzan. Martinus Nijhoff Publishers, Dordrecht, Boston, Lancaster; 237.
Crossref
 

Kong D, Shen H, Li N (2012). Influence of AgNO3 on somatic embryo induction and development in Manchurian ash (Fraxinus mandshurica Rupr.). Afr. J. Biotechnol. 11(1):120-125.

View

 
Kong LS, Yeung EC (1995). Effects of silver nitrate and polyethylene glycol on white spruce (Picea glauca) somatic embryo development: enhancing cotyledonary embryo formation and endogenous ABA content. Physiol. Plant. 93:298-304.
Crossref
 

Kothari-Chajer A, Sharma M, Kachhwaha S, Kothari S (2008). Micronutrient optimization results into highly improved in vitro plant regeneration in kodo (Paspalum scrobiculatum L.) and finger millets (Eleusine coracana (L.) Gaertn.). Plant Cell Tiss. Org. Cult. 94:105-112.

View

 
Lakshmanan P, Taji A (2000). Somatic embryogenesis in leguminous plants. Plant Biol. 2:136-148.
Crossref
 
Louppe D, Oteng-Amoako AA, Brink M (2008). Timbers 1. Plant Resources of Tropical Africa. Wageningen, the Netherlands. PROTA Foundation. Backhuys Publishers. CTA.
 
Mandal AKA, Dutta Gupta S, Chatterji AK (2001). Factors affecting somatic embryogenesis from cotyledonary explants of safflower. Biol. Plantarum. 44:503-507.
Crossref
 
Miche L, Balandreau J (2001). Effects of rice seeds sterilization with Hypochlorite on inoculated Burkholderia vietnamiensis. Appl. Environ. Microbiol. 67(7): 3046-3052.
Crossref
 
Mihaljević I, Dugalić K, Tomaš V, Viljevac M, Pranjić A, ÄŒmelik Z, Puškar B, Jurković Z. (2013). In vitro sterilization procedures for micropropagation of 'OBLAÄŒINSKA' sour cherry. J. Agric. Sci. 58 (20): 117-126.
 
Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassay with tobacco tissue cultures. Plant Physiol. 15:473-497.
Crossref
 

Neondo J, Machua J, Muigai A, Nyende AB, Munjuga M, Jamnadass R, Muchugi A (2011). Micropropagation of Allanblackia stuhlmanii: Amenability to tissue culture technique. Int. J. Biotechnol. Mol. Biol. Res. 2(11):185-194.

View

 
Nieves MC, Evalour TA (2011). Callus Induction in Cotyledons of Moringa oleifera Lam. Phillip. Agric. Sci. 94(3):239-247.
 
Okudera Y, Ito M (2009). Production of agarwood fragrant constituents in Aquilaria calli and cell suspension cultures. Plant Biotechnol. 26:307-315.
Crossref
 

Orwa C, Mutua A, Kindt R, Jamnadass R, Anthony S (2009). Agroforestree Database: a tree reference and selection guide version 4.0.

View

 
Pullman GS, Namjoshi K, Zhang Y (2003). Somatic embryogenesis in loblolly pine (Pinus taeda L.): Improving culture initiation with abscisic acid, silver nitrate, and cytokinin adjustments. Plant Cell Rep. 22:85-95.
Crossref
 
Saiprasad GVS (2001). Artificial seeds and their applications. Resonance, 6:39-47.
Crossref
 
Sakhanokho HF, Rajasekaran K, Kelley RY (2009). Somatic embryogenesis in Hedychium bousigonianum. Hort. Sci. 44:1487-1490.
 
Sanoussi A, Ahoton LE, Odjo Th (2012). Propagation of Black Plum (Vitex doniana Sweet) Using Stem and Root Cuttings in the Ecological Conditions of South Benin. Tropicultura, 2:107-112.
 
Sarker KK, Kabir AH, Sharmin SA, Nasrin Z, Alam MF (2007). Improved somatic embryogenesis using L-Asparagine in wheat (Triticum aestivum L.). Sjemenarstvo, 24(3):187-196
 
Shahsavari E (2011). Contribution of sorbitol on regeneration of embryogenic calli in upland rice. Int. J. Agric. Biol. 13 838–840
 
Shanjani PS (2003). Nitrogen effect on callus induction and plant regeneration of juniperus excelsa. Int. J. Agric. Biol. 5(4):419-422.
 
Steinmacher DA, Cangahuala-Inocente GC, Clement CR, Guerra MP (2007). Somatic embryogenesis from peach palm zygotic embryos. In Vitro Cell Dev. Biol. Plant. 43:124-132. 
Crossref
 
Thies E (1995). Principaux ligneux agro forestiers de la Guinée, Zone de transition. Guinée Bissau, Guinée, Côte d'Ivoire, Ghana, Togo, Bénin, Nigéria, Cameroun. Schriftenreihe der, GTZ No 253.
 
Vikrant RA (2002). Somatic embryogenesis from immature and mature embryos of a minor millet Paspalum scrobiculatum L. Plant Cell Tiss. Org. Cult. 69:71-77.
Crossref
 
Wagstaff SJ, Hickerson L, Spangler R, Reeves PA, Olmstead RG (1998). 'Phylogeny In labiatae S. L., Inferred From Cpdna Sequences'. Plant Syst. Evol. 209(3-4):265-274.
Crossref
 
Webster S, Mitchell SA, Ahmad MH (2003). A novel surface sterilization method for reducing fungal and bacterial contamination of field grown medicinal explants intended for in vitro culture. Proceedings of 17th SRC conference entitled 'Science and Technology for Economic Development: Technology Driven Agriculture and Agro-Processing' SRC, Jamaica.
 
Yan SF, Zhang Q, Wang JE, Sun YQ, Daud MK, Zhu SJ (2010). Somatic embryogenesis and plant regeneration in two wild cotton species belong to G genome. In Vitro Cell Dev. Biol. Plant, 46:298-305.
Crossref
 
Yildiz M, Celal E (2002). The effect of Sodium hypochlorite solution on in vitro seedling growth and shoot regeneration of flax (Linus usitatissimum), Springer-Verlag.
 
Zimmerman JL (1993). Somatic embryogenesis: a model for early development in higher plants. Plant Cell, 5(10):1411-1423. 
Crossref

 




          */?>