Recursos materiales

main problems faced in micropropagation.

Barwale et al. (1986) succeeded in obtain-ing fertile plants in 54 soybean genotypes using callus cultures derived from imma-ture embryos. Plant growth regulators had the greatest impact on the process of callus differentiation. The medium, composed of MS basal salts (Murashige and Skoog, 1962) and B5 vitamins (Gamborg et al., 1968),

10 Micropropagation

E. Skrzypek, I. Czyczyło-Mysza and M. We˛dzony

E. Skrzypek et al.

Arachis correntina L MS NAA, Kin, TDZ Mroginski et al. (2004)

Arachis glabrata L MS NAA, TDZ Vidoz et al. (2004)

Arachis hypogaea L MSB5 BAP, NAA Chengalrayan et al. (1997); Akasaka et al. (2000); Tiwari and Tuli (2009)

Arachis pintoi L, ST MS BAP, NAA, PIC Rey et al. (2000); Rey and Mroginski (2006)

Cajanus cajan CN, L MS IBA, TDZ Singh et al. (2003)

Cicer arietinum CN, EA, N, ST MSB5 BAP, NAA, Kin Sarker et al. (2005); Naz et al. (2007); Rekha and Thiruvengadam (2009)

Glycine max C, CN, EA, H, IE KP8, MS, MSB5, MS ½

2,4-D, BA, BAP, GA_3, IBA, NAA, TDZ

Barwale et al. (1986); Finer and Nagasawa (1988); Dhir et al.

(1992); Bailey et al. (1993); Walker and Parrott (2001); Tomlin et al. (2002); Franklin et al. (2004); Hofmann et al. (2004);

Shan et al. (2005); Radhakrishnan et al. (2009) Lathyrus sativus B, H, ST MSB5 BAP, IAA, NAA, TDZ Zambre et al. (2002); Ochatt et al. (2002)

Lotus corniculatus R Rr, MS BAP Akashi et al. (1998, 2003)

Macrotyloma uniflorum IC MS NAA, Zea, GA3 Mohamed et al. (2005)

Phaseolus acutifolius B MS, B5 TDZ, IAA, BAP Dillen et al. (1996)

Phaseolus coccineus CN, ST MSB5 BAP, NAA, GA₃ Genga and Allavena (1991); Vaquero et al. (1993) Phaseolus vulgaris C, CN, EA MSB5, MS TDZ, BAP Cruz de Carvalho et al. (2000); Veltcheva et al. (2005);

Delgado-Sanchez et al. (2006)

Phaseolus polyanthus C, EA B5 TDZ, IAA Zambre et al. (2001)

Pisum sativum C, CN, E, EA, H, IE, ST

MS, MSB5, KM Pic, Zea, NAA, BAP, 2,4-D, TDZ

Griga (1998, 2000, 2002); Griga et al. (2007); Franklin et al.

(2000); Ochatt et al. (2000); Zhihui et al. (2009)

Vicia faba E, EA, ST MS, KM, SH,

B5

BAP, 2,4-D, NAA, GA₃, Kin, IBA

Skrzypek (2001); Hamdy and Hattori (2006); Bahgat et al.

(2009)

Vigna aconitifolia CN MS 2,4-D, Kin, BA, GA₃ Choudhary et al. (2009)

Vigna mungo CN, ST MS, MS ½ TDZ, NAA Das et al. (1998)

Vigna radiata C, H, L, N MS 2,4-D, IBA, BA, NAA, Kin Devi et al. (2004); Vidoz et al. (2004); Kaviraj et al. (2006) Vigna unguiculata CN, EA, ST MSB5, MS BAP, NAA, IBA Odutayo et al. (2005); Aasim et al. (2009); Raveendar et al. (2009)

aB, vegetative and generative buds; C, cotyledons; CN, cotyledonary nodes; E, epicotyl; EA, embryo axes; H, hypocotyl; IE, immature embryos; L, leaves; N, stem nodes; R, roots; ST, shoot tip.

bB5, Gamborg et al.’s B5 (1968); KM, Kao and Michayluk (1975); MS, Murashige and Skoog (1962); MSB5, Murashige and Skoog with Gamborg’s vitamins (1962); SH, Schenk and Hildebrandt (1972); Rr, Raggio root (Raggio et al. 1957); KP8, (Kao, 1977).

cBAP, 6-benzylaminopurine; BA, benzylamine; 2,4-D, 2,4-dichlorophenoxyacetic acid; GA3, gibberellic acid; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; Kin, kinetin; NAA, 1-naphthaleneacetic acid; Pic, picloram; TDZ, thidiazuron; Zea, zeatin.

Micropropagation 149

was supplemented either by 8 mg/l naphthaleneacetic acid (NAA) or 3 mg/l ben-zylaminopurine (BAP) and 0.037 mg/l NAA.

Either somatic embryogenesis or callusing and organogenesis were achieved. Embryos were converted into plants on the medium supplemented with 0.38 mg/l BAP and 0.04 mg/l indol-3-butyric acid (IBA), while shoot elongation was achieved on media sup-plemented by 1.13 mg/l BAP, 2 mg/l IBA and 1.73 mg/l GA3. Rooting media were based on MS salts without growth regulators. The proper sequence of growth regulators in sub-sequent media is responsible for the success of the procedure, and thus parts of different protocols cannot be combined without careful consideration. The type of explant should be taken into account, since it has a key impact on endogenous phytohormone levels. Here, and in many other leguminous protocols, immature embryos or their parts were used.

The next breakthrough in soybean was reported by Finer and Nagasawa (1988), who elaborated the suspension culture system based on a high level of synthetic auxin ana-logue 2,4-D in the induction medium. Their protocol was applied for soybean transfor-mation (Finer and McMullen, 1991; Trick and Finer, 1998; Santarem and Finer, 1999) and in vitro mutagenesis (Van et al., 2008). Bailey et al.

(1993) made further improvements to the pro-tocol, testing additional growth regulators, source of carbohydrates and other medium additives. Plant recovery was improved via further modifications (Walker and Parrott, 2001; Tomlin et al., 2002; Schmidt et al., 2005).

The latter authors found maltose superior to routinely used sucrose in the conversion rate of embryo to plant. Interestingly, seed pre-treatment with thidiazuron (TDZ) and its addition to the medium in multiple pas-sages enabled longer maintenance of callus tissue without lowering its potential for shoot regeneration (Shan et al., 2005).

Yang et al. (2009), working on a large genotype spectrum, found that the addition of 5 mg/l abscisic acid to the regeneration medium beneficial for embryo conversion to plants. The effect was, however, genotype dependent – genotype was reported to influ-ence the protocol’s efficiency whenever this aspect was studied (Barwale et al., 1986; Parrott

et al., 1989; Dhir et al., 1992; Bailey et al., 1993;

Walker and Parrott, 2001; Tomlin et al., 2002;

Van et al., 2008). Dan and Reighceri (1998) and Reichert et al. (2003) found that the method of utilizing adventitious shoots induced from hypocotyl sections of 7-day-old seedlings was relatively less genotype dependent. Song et al.

(2010) found six QTL associated with somatic embryogenesis that provided potential for marker-assistant selection of genotypes with higher in vitro potential.

10.3 Groundnut (Arachis hypogaea L.)

Arachis hypogaea L. cultivars are known to be relatively recalcitrant to plant regen-eration. Successful results were achieved via organogenesis (Daimon and Mii, 1991;

McKently et al., 1991; Cheng et al., 1992, 1996;

Kanyand et al., 1994; Chengalrayan et al., 1995;

Akasaka et al., 2000; Tiwari and Tuli, 2009) and somatic embryogenesis (Sellars et al., 1990; Durham and Parrott, 1992; Eapen et al., 1993; Chengalrayan et al., 1994, 1997; Baker et al., 1995; Murthy et al., 1995, Joshi et al., 2003). Similar to soybean, a strong influence of genotype was reported (McKently et al., 1990; Matand and Prakash, 2007).

Growth regulators and the type of explant are the key factors for groundnut regenera-tion. Thidiazuron (TDZ) is applied most fre-quently at the start of the culture (Gill and Saxena, 1992; Kanyand et al., 1994; Li et al., 1994; Murthy et al., 1995; Akasaka et al., 2000;

Joshi et al., 2003; Matand and Prakash, 2007), while BAP (6-benzylaminopurine) alone or in combination with NAA (1-naphthalene-acetic acid) is also efficient (Chengalrayan et al., 1995; Akasaka et al., 2000; Banerjee et al., 2007). The immature leaflets isolated from young seedlings are most widely used as explants (Cheng et al., 1992; Chengalrayan et al., 1995, 1997, Akasaka et al., 2000; Joshi et al., 2003; Mroginski et al., 2004; Vidoz et al., 2004; Tiwari and Tuli, 2009). However, peti-oles, mature or immature embryos or their parts and the whole seed were efficient in protocols involving shoot regeneration (Ozias-Akins, 1989; McKently et al., 1990;

Cheng et al., 1992; Gill and Saxena, 1992;

Kanyand et al., 1994; Radhakrishnan et al., 2000; Vasanth et al., 2006). Multiple shoots were induced by Radhakrishnan et al. (2000) from de-embryonated cotyledons, embryo axes and whole mature seeds on MS medium supplemented with BAP. Significant progress in shoot induction rate was claimed in a report by Akasaka et al. (2000). Treatment of 10 mg/l TDZ for 7 days or 1 mg/l TDZ for 21 days was applied to reduce abnormalities in shoot development.

Tiwari and Tuli (2009) obtained excel-lent results for shoot bud formation (85.1%) and shoot elongation (6.2 shoots/explant) when immature leaflets were pre-incubated for 7 days on a medium containing 3 mg/l BAP and 0.92 mg/l NAA. Li et al. (1994) and Tiwari and Tuli (2008) did not observe significant variations in response among cultivated groundnut varieties, similar to the reports of Matand and Prakash (2007).

Somatic embryogenesis was induced in leaf-lets by Narasimhulu and Reddy (1983) and Chengalrayan et al. (1995). Globular embryo-like structures appeared on the cut leaf base on MS medium with 20 mg/l 2,4-D. A high frequency of recovery was found after trans-fer to a medium with 3 mg/l 2,4-D within 20 days, and subsequent culture on that medium with 0.5 mg/l BAP and kinetin (Kin).

Micropropagation and in vitro conservation of wild Arachis species considered as potential sources of novel genes for crop improvement was reviewed by Pacheco et al. (2009).

10.4 Phaseolum (Phaseolus sp.) Plant regeneration in Phaseolus sp. L. was reviewed by Nagl et al. (1997) and Veltcheva et al. (2005). Successful regeneration is reported mainly for Phaseolus vulgaris L. (Benedicic et al., 1991; Malik and Saxena, 1991; Santalla et al., 1998; Cruz de Calvalho et al., 2000).

Regeneration from other Phaseolus species was achieved in Phaseolus coccineus L. (Rubluo and Kartha, 1985; Angelini and Allavena, 1989;

Genga and Allavena, 1991; Malik and Saxena, 1992; Santalla et al., 1998), Phaseolus acutifolius (Dillen et al., 1996; Zambre et al., 1998) and Phaseolus polyanthus (Zambre et al., 2001).

Organogenesis via shoot apex cultures was described by Kartha et al., (1981) and Martins and Sondahl (1984). Cotyledonary nodes and primary leaves were used by McClean and Grafton (1989), Mohamed et al.

(1992) and Vaquero et al. (1993). Axillary meri-stems or shoot apical merimeri-stems (Kartha et al., 1981; Martins and Sondahl, 1984; Rubluo and Kartha, 1985; McClean and Grafton, 1989) were replaced by cotyledons, cotyledonary nodes or the embryonic axis (Mohamed et al., 1992; Santalla et al., 1998).

An enhanced differentiation of somatic embryos in cotyledonary leaf-derived cal-lus but low regeneration frequency has been reported for P. vulgaris L. by Mohamed et al.

(1993). A high frequency of direct shoot for-mation from intact seedlings has been estab-lished by Malik and Saxena (1992) using TDZ and BAP, while seedling-derived thin layers were used to improve regeneration (Cruz de Carvalho et al., 2000). The latter group reported successful development of shoots from bud primordia on a medium with TDZ and AgNO3, with a high rate of develop-ment of fertile plants. A protocol based on embryo-axes derived from mature seeds was reported by Delgado-Sanchez et al. (2006). All results cited above point to strong genotype dependence and lack of universal protocol for Phaseolus species.

10.5 Pea (Pisum sativum L.)