African Journal of
Agricultural Research

  • Abbreviation: Afr. J. Agric. Res.
  • Language: English
  • ISSN: 1991-637X
  • DOI: 10.5897/AJAR
  • Start Year: 2006
  • Published Articles: 6688

Full Length Research Paper

Embryo-like structures and root regeneration induced by 2, 4-dichlorophenoxyacetic acid in twenty African sweet potato cultivars

Abel Sefasi
  • Abel Sefasi
  • School of Agricultural Sciences, Makerere University, Box 7062, Kampala, Uganda.
  • Google Scholar
Marc Ghislain
  • Marc Ghislain
  • International Potato Center, Box 1558, Lima 12, Peru.
  • Google Scholar
Andrew Kiggundu
  • Andrew Kiggundu
  • National Agricultural Research Laboratories Institute, Kawanda, P. O. Box 7065, Kampala, Uganda.
  • Google Scholar
Gorrettie Ssemakula
  • Gorrettie Ssemakula
  • National Crops Resources Research Institute, Box 7084, Kampala, Uganda.
  • Google Scholar
Runyararo Rukarwa
  • Runyararo Rukarwa
  • School of Agricultural Sciences, Makerere University, Box 7062, Kampala, Uganda.
  • Google Scholar
Robert Mwanga
  • Robert Mwanga
  • International Potato Center, Box 22274, Kampala, Uganda.
  • Google Scholar
Jan Kreuze
  • Jan Kreuze
  • International Potato Center, Box 1558, Lima 12, Peru.
  • Google Scholar
Settumba Mukasa
  • Settumba Mukasa
  • School of Agricultural Sciences, Makerere University, Box 7062, Kampala, Uganda.
  • Google Scholar


  •  Received: 11 April 2016
  •  Accepted: 24 June 2016
  •  Published: 06 April 2017

 ABSTRACT

Embryo-like structures were induced from ‘whole leaf’ explants of sixteen of the twenty investigated sweet potato cultivars on Murashige and Skoog (1962) medium supplemented with 2,4 Dichlorophenoxyacetic acid (0.2 mg/L) at the Makerere University Agricultural Research Institute Tissue culture laboratory. Shoot and root regeneration was possible in the non-African cultivar, Jonathan, whereas only root regeneration was successfully induced in five African cultivars. Cultivar type had a highly significant (P < 0.001) effect on frequency of embryo-like structures and efficiency of both shoot and root regeneration. The embryo-like structures induced could be useful for initiation of cell suspensions to enable genetic transformation of African cultivars. The method for de novo regeneration of roots has potential application in the regeneration of plants or hairy root cultures for cultivars that are recalcitrant to shoot regeneration.

 

Key words: 2, 4-dichlorophenoxyacetic acid, cultivar, in vitro recalcitrance, regeneration efficiency, somatic embryogenesis.


 INTRODUCTION

Sweet potato (Ipomoea batatas (L.) Lam.) is mainly cultivated for its expanded edible roots which contain high  carbohydrate   and   beta-carotene,   depending   on cultivar. The total sweet potato production for the world is 103.1 million metric tons (USDA, 2015). However, the potential   production   for   sub-Saharan   Africa   is    not attained, as yields are very low at about 5 t/ha (Luo et al., 2006). The low yields are mainly due to various challenges including high incidence of virus disease and increasing attacks by sweet potato weevils (Fuglie, 2007).
 
For a long time, traditional breeding has contributed to the improvement of crop traits. Nevertheless, due to the high male sterility, self and interspecific incompatibility and hexaploid (2n = 6x = 96 chromosomes) nature of sweet potato, conventional hybridization is still difficult, and hybrid progenies undergo segregation leading to loss of many valuable traits (Yang et al., 2011). Genetic transformation is a promising tool to improve sweet potato traits like nutrition and resistance to weevils and viruses (Ghislain et al., 2008; Kreuze et al., 2008; Shimada et al., 2006). Genetic transformation enables access to genes from unrelated species thereby enabling breeders to improve plants with traits that are not available in the crop germplasm (Ghislain et al., 2008).
 
Efficient development of transgenic plants requires optimal transformation and regeneration conditions. Although, successful induction of somatic embryogenesis has been reported in sweet potato, most of the published protocols are cultivar-specific (Santa-Maria et al., 2009). Low embryogenic frequencies and complete recalcitrance has been reported for most important and adapted sweet potato cultivars (Al-Mazrooei et al., 1997; Liu et al., 2001; Zang et al., 2009), particularly for African cultivars (Luo et al., 2006). The non-African cultivars that have been transformed so far, including Jewel (Luo et al., 2006), Huachano (Kreuze et al., 2008), White Star (Gama et al., 1996), Kokei 14 (Otani et al., 1998), Yulmi (Choi et al., 2007) and Lizixiang (Yu et al., 2007), can only be used for research purposes in Africa. These are not farmer-preferred cultivars due to several drawbacks, such as low dry matter and vulnerability to diseases and pests (Yang et al., 2011). Hence, the development of an efficient in vitro regeneration and transformation method for farmer-preferred sweet potato varieties in sub-Saharan Africa is important and urgently required.
 
The objective of the study reported here was to screen a large number of farmer-preferred sweet potato cultivars for the response to somatic embryogenesis induced by 2,4-Dichlorophenoxyacetic acid (2,4-D). Published reports show that there is a correlation between the quality and quantity of embryo-like structures and subsequent plant regeneration in sweet potato (Al-Mazrooei et al., 1997).


 MATERIALS AND METHODS

Twenty farmer-preferred Ugandan sweet potato cultivars and one USA cultivar, Jonathan, were used in this study. These cultivars as shown in Table 1 were selected based on preferred attributes among consumers in Uganda, including high dry matter content (Mwanga et al., 2007; Mwanga et al., 2001). The non-African cultivar, Jonathan, was previously reported to regenerate through somatic embryogenesis (Cipriani et al.,  1999).  The  cultivars  were grown in a screenhouse at Makerere University Agricultural Research Institute, Kabanyolo. Vines from the screen house grown plants were grafted on Ipomoea setosa, an indicator plant for virus infection (Figure 1). Cuttings from those sweet potato plants corresponding to scions whose indicator plants were asymptomatic were used in establishment of virus-free in vitro cultures.
 
 
 
 
 
Cuttings containing five to eight nodes were taken from the greenhouse to the laboratory. The cuttings were washed for fifteen minutes with flowing tap water and then immersed for 20 min in 39% (v/v) commercial JIK containing 3.85 % sodium hypochlorite (NaOCl) and 0.03% Tween 20®. Thereafter the vines were submerged in 70% ethanol for 2 min. After surface sterilization, the vines were transferred to sterile water and rinsed three times. They were then each cut into one or two nodes and inserted into sweet potato propagation medium.
 
The propagation medium was composed of MS (Murashige and Skoog, 1962) salts premix (4.3 g l-1), sucrose (30 g/L), myo-inositol (0.1 g/L), 5 ml/L sweet potato vitamin stock comprised of 40 g/L ascorbic acid, 20 g/L L-arginine, 4 g/L putrescine HCl, 0.01 mg/L gibberellic acid (GA3) and 0.4 g/L calcium pantothenate (Kreuze et al., 2008). All reagents used in this study were sourced from Sigma-Aldrich. The medium was adjusted to pH 5.8 before adding 3 g/L phytagel and autoclaved at 121°C for 15 min under 15 kPa.
 
Four-week-old whole leaves (petiole with lamina) from the in vitro-grown plants were placed on callus induction medium (CIM) under dark conditions at 28°C. The cut end of the petiole was slightly pushed into medium. CIM was made of 4.3 g/L MS premix (without vitamins), 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 1 ml/L Vitamins stock (0.5 mg/ml nicotinic acid, 0.1 mg/ml thiamine and 0.5 mg/ml pyridoxine), 100 mg/L myo-inositol, 30 g/L sucrose and 7 g/L agar. The cultures were transferred to fresh media every two weeks. A total of ten to fifteen explants were used per cultivar in each petri dish, and this was replicated three times and arranged in a completely randomized design. After twelve weeks the callus formed was transferred to embryo initiation medium (EIM) under a photoperiod of 16 h, regardless of it being embryogenic or non-embryogenic. EIM was made as CIM but 2, 4-D was replaced with 1 mg/L abscisic acid (ABA). After four weeks on EIM, the callus was transferred to plant regeneration medium devoid of plant growth regulators. Fully developed plants were transferred to soil in a screen house where they developed normally.
 
Data on number of explants forming callus and those forming embryo-like structures were collected after twelve weeks. Data on shoot and root regeneration was collected every week since some callus formed roots within three weeks of culture initiation. Cultures were discarded after twenty-four weeks since at this stage most calli lost their embryogenic capacity and could not regenerate shoots. Frequency data was transformed using the arcsine square root before analysis to stabilize the variance. The table presented in this paper shows non-transformed data expressed as percentages. GenStat statistical package was used to analyse the effect of cultivar on total callus proliferation and formation of embryo-like structures, which was performed using general linear model of analysis of variance (ANOVA) at 5% level of significance. Mean values were compared using the least significance difference (LSD) method at the 5% level.


 RESULTS

In the present study, explants placed on callus induction medium responded by producing callus within three days. The callus was not only on the wounded tip of the petiole but also on the intact leaf lamina (Figure 2B). Two types of callus were induced; embryogenic and non-embryogenic.  The  embryo-like  structures   were   bright yellow or orange in colour and compact in appearance, while non-embryogenic callus was white and friable and proliferated earlier and more rapidly than embryo-like structures. The embryo-like structures were induced in sixteen of the twenty investigated African cultivars cultured on media with 0.2 mg/L 2,4-D. Cultivar type had a highly significant (P < 0.001) effect on embryo-like structures induced (Table 2). The frequency of embryo-like structures ranged from zero to 25% depending on cultivar (Table 2). Embryo-like structures started developing within six weeks of initiation of cultures.
 
 
 
Somatic embryos of cv. Jonathan developed into shoots after three weeks on ABA-containing media. Cultivar type also had a highly significant (P < 0.001) effect on the ability to regenerate shoots (Table 2). Regeneration of roots was achieved from callus of five African cultivars. Contrary to the regeneration of shoots which occurred through somatic embryogenesis, the regeneration of roots appeared to be so through organogenesis as they were formed directly from callus without somatic embryos. In addition, the roots were regenerated earlier than shoots, sometimes, as early as three weeks after culture initiation on 2, 4-D-containing media. The emergence of new roots was sustained throughout the culture period. The type of cultivar showed a highly significant (P < 0.001) effect on the ability to regenerate roots (Table 2).


 DISCUSSION

Sweet potato has for long been considered recalcitrant to somatic embryogenesis (Yang et al., 2011). Most of the reported protocols are cultivar specific, and have low regeneration frequencies which are also difficult to reproduce (Santa-Maria et al., 2009; Yang et al., 2011; Yu et al., 2007). In the present study, the auxin 2, 4-D was used for callus induction and embryo-like structures was achieved in sixteen of the investigated twenty African cultivars. These cultivars have not been reported to respond to somatic embryogenesis in previous publications. Luo et al. (2006) reported that African cultivars were difficult to regenerate, but did not name the cultivars tested or their geographical location.
 
The  induction  of  somatic  embryogenesis  with  2, 4-D appears to be preferable in view of existing literature (Yang et al., 2011). Liu et al. (2001) induced embryo-like structures from shoot apices of fifteen cultivars from China and Japan on medium supplemented with 9.05 mM 2, 4-D. The embryogenic response of these cultivars was low and so variable ranging from 6.7 to 85.2% (Liu et al., 2001). Similarly, Sihachakr et al. (1997) found large differences and low frequency of embryo-like structures among ten sweet potato cultivars exposed to auxin (10 µM 2, 4-D) treatment. Frequencies ranged from 0 to 17% response when using lateral buds as explants (Sihachakr et al., 1997). The frequency of embryo-like structures in the current study ranged from no response at all to 25% depending on cultivar (Table 2). In contrast to somatic embryogenesis protocols, most auxin-cytokinin shoot organogenesis protocols have replaced 2, 4-D with other auxins in order to avoid the callus stage and achieve rapid regeneration (Luo et al., 2006, Gong et al., 2005; Santa-Maria et al., 2009).
 
Although, somatic embryogenesis is a complicated process to manage than shoot organogenesis (Luo et al., 2006; Newell et al., 1995), the regeneration of sweet potato through somatic embryogenesis is still a preferred option than shoot organogenesis (Yu et al., 2007). This is mainly because somatic embryogenesis leads to high efficiency of selection of transgenic plants on medium and avoids the regeneration of chimeric plants after genetic transformation (Song et al., 2004; Yang et al., 2011). Recently, there have been some reports of applying somatic embryogenesis using cell suspensions (Yu et al., 2007; Zang et al., 2009). However, other reports suggest that the use of liquid cultures is extremely complicated and depends on availability of cell suspension cultures (Song et al., 2004; Zhai and Liu, 2003). By opting for solid medium in this study, it was possible to deploy explants which are easy to access and are readily available as noted in previous reports (Cipriani et al., 1999; Kreuze et al., 2008; Song et al., 2004). The use of solid
 
medium for somatic embryogenesis has allowed many researchers to use various plant organs as explants for example, leaf discs (Newell et al., 1995), stem internodes (Song et al., 2004), root explants (Liu and Cantliffe, 1984), root discs (Newell et al., 1995) and lateral buds (Cavalcante Alves et al., 1994).
 
The regeneration of roots in this work could be useful for the initiation of root cultures, which could serve as a source of tissue for regeneration of plants or hairy root cultures (Jones et al., 2007; Dodds et al., 1991). However, it remains difficult to explain this type of morphogenesis which was evident as early as three weeks after culture initiation on 2, 4-D-supplemented medium. It is likely that the explants of the cultivars had high levels of endogenous auxins. If this is the case then the exogeneous auxin 2, 4-D only accelerated the root regeneration process (Becerra et al., 2004; George et al., 2008). Many factors have been implicated in the unpredictable responses in culture, including variations in the  developmental  and  physiological  stage  of  in   vitro plants (Triqui et al., 2007). Previous reports indicate that ABA is required for induction, maturation and synchronization of somatic embryos leading to elongation into shoots (Anwar et al., 2010; Song et al., 2004; Triqui et al., 2007). However, in the current study, two Ugandan cultivars, Magabali and Kyebandula that induced more embryo-like structures than the embryogenic callus reported for the USA cultivar, Jonathan, did not regenerate shoots. The failure of ABA to induce somatic embryos or promote development of preformed somatic embryos in the sixteen African cultivars that successfully induced embryo-like structures could be attributed to cultivar differences in the response to ABA or the sustained effect of  2,4-D  as  reported  previously (Becerra et al., 2004; George et al., 2008; Santa-Maria et al., 2009).
 
In conclusion, this study has identified up to sixteen African sweet potato cultivars which were able to induce embryo-like structures on medium supplemented with the potent auxin 2,4-D (0.2 mg/L). The embryo-like structures induced in this study could be useful for the initiation of embryogenic cell suspensions which are not readily available for transformation of most sweet potato cultivars (Yang et al., 2011).The method for de novo regeneration of roots may be applicable in the regeneration of plants or hairy root cultures. A possibility exists that the use of other types of plant organs as explants and various concentrations of 2, 4-D or other auxins could improve somatic embryogenesis in the cultivars investigated. This view is now being pursued as a follow-up study. The final goal of this work is to use the somatic embryogenesis protocol for Agrobacterium-mediated transformation in order to improve sweet potato traits.


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.



 REFERENCES

Al-Mazrooei S, Bhatti MH, Henshaw GG, Taylor NJ, Blakesley D (1997). Optimisation of somatic embryogenesis in fourteen cultivars of sweetpotato [Ipomoea batatas (L.) Lam.]. Plant Cell Reports 16:710-714.
Crossref

 

Anwar N, Watanabe KN, Watanabe JA (2010). Transgenic sweet potato expressing mammalian cytochrome P450. Plant Cell Tis. Organ Cult. 105:219-231.
Crossref

 
 

Becerra DC, Forero AP, G’ongora GA (2004). Age and physiological condition of donor plants affect in vitro morphogenesis in leaf explants of Passiflora edulis f. flavicarpa. Plant Cell Tiss. Organ Cult. 79:87-90.
Crossref

 
 

Cavalcante-Alves JM, Sihachakr D, Tizroutine S, Mussio L, Servnes A, Ducreux G (1994). Isozyme modification and plant regeneration through somatic embryogenesis in sweet potato (Ipomoea batatas (L.) Lam.). Plant Cell Reports 13:437-441.
Crossref

 
 

Choi HJ, Chandrasekhar T, Lee HY, Kim KM (2007). Production of herbicide resistant transgenic sweetpotato plants through Agrobacterium tumefaciens method. Plant Cell Tis. Organ Cult. 91:235–242.
Crossref

 
 

Cipriani G, Michaud D, Brunelle F, Golmirzaie A, Zhang DP (1999). Expression of soybean proteinase inhibitor in sweetpotato. Impact on a Changing World. International Potato Center, Lima pp. 271-277.

 
 

Dodds JH, Merzdorf C, Zambrano V, Sig¨ue˜nas C, Jaynes J (1991). Potential use of agrobacterium-mediated gene transfer to confer insect resistance in sweetpotato. In: Sweetpotato pest management: A global perspective, R.K. Jansson and K.V. Raman (Eds), West view Press, Oxford, UK. pp. 203-219.

 
 

Fuglie KO (2007). Priorities for sweetpotato research in developing Countries: Results of a survey. HortScience 42:1200-1206.

 
 

Gama ICSM, Leite RP, Cordeiro AR, Cantliffe DJ (1996). Transgenic sweet potato plants obtained by Agrobacterium tumefaciens-mediated transformation. Plant Cell Tis. Organ Cult. 46:237-244.
Crossref

 
 

George EF, Hall MA, de Klerk GJ (2008). (Eds.) Plant propagation by tissue culture 3rd edition pp. 335-354.

 
 

Ghislain M, Moar WJ, Mwanga ROM, Tovar J, Prentice K, Ormachea M, Rivera C, Manrique S, Wamalwa L, Ekobu M, Solera M (2008). Progress towards resistance to African sweetpotato weevils using biotechnology. In Book of Abstracts of the First All Africa Biotechnology Congress, 22nd September 2008, Nairobi, Kenya. P 26.

 
 

Gong Y, Gao F, Tang K (2005). In vitro high frequency direct root and shoot regeneration in sweet potato using the ethylene inhibitor silver nitrate. S. Afr. J. Bot. 71:110-113.
Crossref

 
 

Jones MPA, Yi Z, Murch SJ, Saxena PK (2007). Thidiazuron-induced regeneration of Echinacea purpurea L.: Micropropagation in solid and liquid culture systems. Plant Cell Reports 16:13-19.

 
 

Kreuze JF, Klein IS, Lazaro MU, Chuquiyuri WC, Morgan GL, Mejía PGC, Ghislain M, Valkonen JPT (2008). RNA silencing-mediumted resistance to a crinivirus (Closteroviridae) in cultivated sweetpotato (Ipomoea batatas L.) and development of sweetpotato virus disease following co-infection with a potyvirus. Molec. Plant Pathol. 9:589-598.
Crossref

 
 

Liu JR, Cantliffe DJ (1984). Somatic embryogenesis and plant regeneration in tissue culture of sweet potato (Ipomoea batatas Pois.). Plant cell Reports 3:112-112.
Crossref

 
 

Liu QC, Zhai H, Wakita Y, Zhang DP (2001). Efficient plant regeneration from embryogenic suspension cultures of sweetpotato. In vitro cell Development Biology-Plant 37:564-567.

 
 

Luo HR, Santa-Maria M, Benavides J, Zhang DP, Zhang YZ, Ghislain M (2006). Rapid genetic transformation of sweetpotato (Ipomoea batatas (L.) Lam) via organogenesis. Afr. J. Biotechnol. 5:1851-1857.

 
 

Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant. 15:473-497.
Crossref

 
 

Mwanga ROM, Odongo B, Alajo A, Kigozi B, Niringiye C, Kapinga R, Tumwegamire S, Makumbi R, Lugwana E, Namakula J, Lemaga B, Nsumba J, Yencho C (2007). Submission to the Variety Release Committee for the release of sweetpotato varieties. National Agricultural Research Organization (NARO) National Crops Resources Research Institute (NaCRRI), Kampala, Uganda. ISBN 9970-895-11-1. 58 p.

 
 

Mwanga ROM, Odongo B, Ocitti p'Obwoya C (2001). Release of Five Sweetpotato Cultivars in Uganda. Hortscience 36:385-386.

 
 

Newell CA, Lowe JM, Merryweather A, Rooke LM, Hamilton WDO (1995). Transformation of sweet potato (Ipomoea batatas (L.) Lam.) with Agrobacterium tumefaciens and regeneration of plants expressing cowpea trypsin inhibitor and snowdrop lectin. Plant Sci. 107:215-227.
Crossref

 
 

Otani M, Shimada T, Kimura T, Saito A (1998). Transgenic plant production from embryogenic callus of sweetpotato (Ipomoea batatas (L.) Lam.) using Agrobacterium tumefaciens. Plant Biotechnol. 15:11-16.
Crossref

 
 

Santa-Maria M, Pecota KV, Yencho CG, Allen G, Sosinski B (2009). Rapid shoot regeneration in industrial 'high starch' sweetpotato (Ipomoea batatas L.) genotypes. Plant Cell Tis. Organ Cult. 109:109-117.
Crossref

 
 

Shimada T, Otani M, Hamada T, Kim SH (2006). Increase of amylose content of sweetpotato starch by RNA interference of the starch branching enzyme II gene (IbSBEII). Plant Biotechnol. 23:85-90.
Crossref

 
 

Sihachakr D, Haicour R, Cavalcante Alves JM, Umboh I, Nzohge D, Servaes A, Ducreaux G (1997). Plant Regeneration in sweetpotato (Ipomoea batatas L., Convolvulaceae). Euphytica 96:143-152.
Crossref

 
 

Song GQ, Honda H, Yamaguchi KI (2004). Efficient Agrobacterium tumefaciens–mediated transformation of sweet potato (Ipomoea batatas (L.) Lam.) from stem explants using a two-step kanamycin-hygromycin selection method. In vitro Cell Dev. Plant 40:359-365.

 
 

Triqui ZEA, Guédira A, Chlyah A, Chlyah H, Souvannavong V, Haïcour R, Sihachakr D (2007). Effect of genotype, gelling agent, and auxin on the induction of somatic embryogenesis in sweet potato (Ipomoea batatas Lam.). Plant Biol. Pathol. 331:198-205.

 
 

United States Department of Agriculture (USDA) (2015). Economic Research Services. Situation and Outlook. VGS-355-SA1.

 
 

Yang J, Bi HP, Fan WJ, Zhang M, Wang HX, Zhang P (2011). Efficient embryogenic suspension culturing and rapid transformation of a range of elite genotypes of sweet potato (Ipomoea batatas [L.] Lam.). Plant Sci. 181:701-711.
Crossref

 
 

Yu B, Zhai H, Wang Y, Zang N, He S, Liu Q (2007). Efficient Agrobacterium tumefaciens-mediumted transformation using embryogenic suspension cultures in sweetpotato, Ipomoea batatas (L.) Lam. Plant Cell Tis. Organ Cult. 90:265-273.
Crossref

 
 

Zang N, Zhai H, Gao S, Chen W, He S, Liu Q (2009). Efficient production of transgenic plants using bar gene for herbicide resistance in sweetpotato. Short Communication. Sci. Horticult. 122:649-653.
Crossref

 
 

Zhai H, Liu QC (2003). Studies on the genetic transformation of embryogenic suspension cultures in sweet potato. Sci. Agric. Sin. 36:487-491.

 

 




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