callus was published by Gupta et al. in 1972 and on soybean by Tang et al. (1973) but, after over 30 years, haploid protocols are still not routinely used in any food legume breeding programme. However, recent progress in pea (Ochatt et al., 2009) and chickpea (Grewal et al., 2009) androgenesis, through combination of various stresses (cold, electroporation, centri-fugation and osmotic shock), suggests that this recalcitrance can be overcome in the Fabaceae.
Table 11.1 lists the androgenesis studies con-ducted during over the years in different food legume species, and these are discussed below in detail.
Field pea (Pisum sativum L. subsp.
sativum var. arvense)
Since the onset of genetic studies with plants, pea (2n = 14) has been a preferred species for study. In terms of haploid development, this was also true with the first report on hap-loid callus induction of anthers (Gupta et al., 1972), and with the recovery of a few hap-loid pea plants (Gupta 1975), although these results could not be reproduced subsequently (Table 11.1). Using cold treatment for 72 h, Gosal and Bajaj (1988) obtained 0.34% embry-oid formation. Croser and Lulsdorf (2004) tested cold or heat stress for the induction of microspores resulting in symmetrical micro-spore nuclei division. Recent research under-lined the difficulty of producing confirmed haploids, with most results stopping short of the recovery of pea plants (Croser et al., 2005, 2007; Sidhu and Davies, 2005), irrespective of the use of male organs (anthers) or reduced
gametophytes (microspores) as starting ma -terial. Recently, a small number of plants were recovered from isolated microspores of a few field pea genotypes (Ochatt et al., 2009). Thus, five plants were obtained through organogen-esis from microspore-derived calli (one from cv. Victor and four from cv. Frisson), and three more plants were produced via embryogen-esis from the microspores of cv. CDC April (Table 11.1).
Chickpea (Cicer arietinum L.) Khan and Ghosh (1983) were the first to report in vitro androgenesis in chickpea (2n = 14).
Three pollen embryoids were regenerated from calli, but plants were not obtained. Altaf and Ahmad (1986) used a cold pre-treatment of buds at 4°C for 3–7 days and centrifugation for 45 min at 1000 RPM, resulting in callus devel-opment from the anthers. However, shoots could not be obtained and the ploidy status of the callus cells was not determined. Bajaj and Gosal (1987) induced callus from anthers cold-treated for 3 days, on MS medium with various hormones; a few multicellular embryoids were obtained. Later, Huda et al. (2001) found that cold treatment of anthers and a B5 (Gamborg et al., 1968) medium with either 2,4-D or NAA was suitable for induction of androgenesis.
After callus induction, a few embryos and shoots developed, but ploidy level was not determined. Mature embryos were obtained by Vessal et al. (2002), using cold treatment of buds for 7–10 days, followed by anther cul-ture on MS medium with 1 mg/l 2,4-D and 0.2 mg/l kinetin. Embryos were regenerated from haploid callus on a modified Blaydes’
(1966) medium with 0.5 mg/l kinetin and 10% sucrose. Callus growth consisted of cells with haploid to polyploid chromosome num-bers. Similarly, Croser et al. (2005) used iso-lated microspore culture and a modified MS medium to obtain androgenesis in three chick-pea cultivars (Table 11.1).
The first confirmed haploid plants from anther culture were reported by Grewal et al.
(2009) for cv. CDC Xena (kabuli) and cv. Sonali (desi) (Table 11.1). Induction required a four-step stress treatment consisting of: (i) a 72 h cold treatment of buds; (ii) centrifugation (168 g)
Androgenesis and Doubled-haploid Production 161 Table 11.1. Overview of target explants, stresses and media used for induction of androgenesis in food legume species.
Target explantsaStress sequence Mediumb Reference(s)
Pea
A – I: White + 2,4-D + coconut milk Gupta et al. (1972)
A – I: White + NAA + coconut milk Gupta (1975)
A Cold for 72 h (A) I: Various MS-based media Gosal and Bajaj (1988)
M Cold or heat (buds) I: Various semi-solid media with 2,4-D; S: hormone-free medium Croser and Lulsdorf (2004) M Cold (buds) I: Modified ML6 + 1 mg/l NAA + 15% fructose maltose, or 9% sucrose Croser et al. (2005, 2007) A Cold for 72 h (A) I: B5 + 2 mg/l Dicamba + 300 mg/l casein hydrolysate + 9% sucrose; S: ELS
on L2 + 1 mg/lBAP + 2% sucrose
Sidhu and Davies (2005)
M a) Cold > 48 h (buds) b) Electroporation
I: Liquid stationary culture on NLN or HSO, 1 month S: Same media but semi-solid
Ochatt et al. (2009)
Chickpea
A I: MS + 2 mg/l2,4-D + 10% coconut milk; S: as I but + 500 mg/l acalbumin
hydrolysate
Khan and Ghosh (1983)
A a) Cold 72–168 h (buds) b) Centrifugation at 1000 RPM for 45 min at 4°C (buds)
I: MS or B5 + 2.21 mg/l 2,4-D + 0.225 mg/l BAP Altaf and Ahmad (1986)
A Cold 72 h (A) I: MS + 4 mg/l IAA + 2 mg/l Kin Bajaj and Gosal (1987)
A Cold 72–168 h (A) I: cv. Nabin on B5 + 2 mg/l 2,4-D + 2 mg/l BAP; I: cv. ICCL83105 on B5 + 2 mg/l NAA + 2 mg/l BAP ; S: B5 + 0.5 mg/l IAA + 1 mg/l BAP + 0.5 mg/l Kin
Huda et al. (2001)
A Cold 168–240 h (buds) I: MS + 1 mg/l 2,4-D + 0.2 mg/l Kin S: Modified Blaydes + 0.5 mg/l Kin + 10%
sucrose
Vessal et al. (2002)
M Cv. Narayen 32.5°C for 16 h Cv.
Sona 48 h cold (buds) Cv.
Rupali none
I: Modified MS + 1 mg/l 2,4-D + 0.25 mg/l Pic + 0.1 mg/l BAP + 9% sucrose
Croser et al. (2005)
A a) Cold 72 h (buds) b) Centrifugation of 168 g for 10 min (anthers) c) Electroporation with 625 V/
cm, 25 μF and 25 Ω (A) d) High osmotic liquid medium for 4 days (A)
I: RM-IK + 4 mg/l IAA + 0.4 mg/l Kin + 17% sucrose S1: Modified L2 + 1 mg/l Pic + 0.40 mg/l 2iP + 4% sucrose + 5% maltose; S2:
Modified L2 + 4 mg/l IAA + 1 mg/l ZR + 5 mg/l GA3 + 1 mg/l ABA; S3:
Modified MS + 0.01 mg/l NAA + 0.1 mg/l BA + 4.5% sucrose + 4.5 % maltose
Grewal et al. (2009)
Continued
M.M. Lulsdorf et al.
Lentil
A, M Heat or cold not effective I: ML6 + 2 mg/l 2,4,5-T + 1 mg/l BAP + 6% sucrose Keller and Ferrie (2002) M Cold 96 h I: Modified R&D + 1mg/l 2,4-D + 1 mg/l NAA + 1 mg/l Kin 10% sucrose Croser and Lulsdorf (2004) Soybean
A I: Miller’s + 20 mg/l NAA + 1 mg/l Kin Ivers et al. (1974)
A I: B5 + 2 mg/l 2,4-D + 12% sucrose Yin et al. (1982)
A I: Modified B5 + 2 mg/l 2,4-D + 2 mg/l BAP + 0.5 mg/l Kin + 12% sucrose Jian et al. (1986) Cold 120–192 h + 2 mg/l 2,4-D
(buds)
I: Enriched B5 + 0.5 - 1.0 mg/l NAA + 0.1- 0.5 mg/l zeatin Liu and Zhao (1986)
A Cold 4–8 days; 37°C for 24 h (buds)
I: B5 ‘long’ + 2 mg/l 2,4-D + 0.5 mg/l BA + 9% sucrose + 0.3% agarose Zhuang et al. (1991)
A Cold 72–120 h (buds) I: Modified MS and B5 + 2 mg/l 2,4-D + 12% sucrose S: B5 + 0.5 mg/l NAA + 1 mg/l Kin + 1% sucrose S: Modified MS + 0.5 mg/l IBA
+ 0.5 mg/l BAP, 0.5 mg/l Kin, O.5 mg/l zeatin + 5% sucrose + 1% maltose
Ye et al. (1994)
A Cold 96 and 192 h or heat (37°C)
I: B5 ‘long’ + 2 mg/l 2,4-D + 0.5 mg/l BAP + 9–12% sucrose + 0.35% agarose
Hu et al. (1996)
A Cold 0–10 days (buds) I: B5 ‘long’+ 2 mg/l 2,4-D + 0.5 mg/l BAP + 9% sucrose + 0.8% agarose Kaltchuk-Santos et al. (1997) A Cold 24–48 h (buds) I: B5 or B5 ‘long’ + YS amino acids + 2 mg/l 2,4-D + 0.5 mg/l BAP + 9%
sucrose + 0.3% phytagel
Cardoso et al. (2004)
A Cold 12 h (buds) I: B5 ‘long’ + YSaa + 2 mg/l 2,4-D + 0.5 mg l−1BAP + 9% sucrose + 0.25%
phytagel; S: as above but 1 mg/l 2,4-D + 1 mg/l BAP; S: MSO: MS salts + B5 vitamins + 3% sucrose + 0.25% phytagel; S: MSO + 1% sucrose
de Moraes et al. (2004)
A Cold 0–10 days (buds) I: B5 ‘long’+ YSaa + 2 mg/l 2,4-D + 0.5 mg/l BAP + 9% sucrose + 0.8%
agarose; S: B5 + 1 mg/l 2,4-D + 3 mg/l BAP + 3% sucrose
Rodrigues et al. (2004a, b)
A Cold 3–5 days (buds) I: B5DBIG + 2 mg/l 2,4-D + 0.5 mg/l IBA + 100 mg/l myo-inositol + 360 mg/l L-glutamine + 9% sucrose + 0.7% agar S: MS + 0.4 mg/l NAA + 0.4 mg/l BAP + 2% sucrose + 0.8% agar
Tiwari et al. (2004)
A I: B5 ‘long’+ YSaa + 2 mg/l 2,4-D + 0.5 mg/l BAP + 9% sucrose
+ 0.25% phytagel
Rodrigues et al. (2005a)
M I: Modified PTA-15 Rodrigues et al. (2006)
A Cold 24–48 h (buds) I: B5 and B5 ‘long’+ YS amino acids + 2 mg/l 2,4-D + 0.5 mg/l BAP + 9%
sucrose + 0.3% phytagel or modified PTA-15
Cardoso et al. (2007)
Androgenesis and Doubled-haploid Production 163 Common bean
A I: B5 + 2 mg/l 2,4-D + 0.2 mg/l Kin + 2% sucrose Haddon and Northcote (1976)
A I: 67V + 1 mg/l 2,4-D (or 1mg/l NAA + 2 mg/l IAA + 0.2 mg/l Kin) +
0.2% casein hydrolysate + 2% sucrose
Peters et al. (1977)
A I: B5 + 2 mg/l 2,4-D + 1 mg/l Kin Tai and Cheng (1990)
A Cold 0–48 h (buds) I: MS + 2 mg/l 2,4-D + 2 mg/l Kin + 0.2% casein hydrolysate + 1.25–5.0%
sucrose or maltose
Muñoz-Florez et al. (1992);
Muñoz et al. (1993);
Muñoz and Baudoin (1994, 2001–2002), Lupin
A I: MS + 1 mg/l 2,4-D + 1 mg/l BAP + 1−1 mg/l IAA or 1.5 mg/l+ 0.2 mg/l NAA or 2 mg l−1BAP + 0.2 mg/l NAA
Sator (1985)
M I: NNB5 + 1 mg/l NAA + 0.5 mg/l 2,4-D + 1 mg/l Kin + 0.5 mg/l BAP + 5%
sucrose or maltose + 0.8 g/l L-proline + 0.1 g/ l L-serine S: S&H + 0.09 mg/l GA3 + 5% sucrose or maltose + 0.8% agar
Ormerod and Caligari (1994)
M a) 6, 22 or 30°C for 24 h, 48 h and 72 h (buds) b) Centrifugation for 15 min at 130 × g then 2 × 5 min at 100 × g (M)
I: N1 = NN basal medium + 36.7 mg/l NaFeEDTA + 13% sucrose + 30 mg/l glutathione + 0.8 g/l glutamine + 0.1 g/l serine S1: N2 = N1 but with 0.1 mg/l IAA + 0.01 mg/l zeatin + 6% sucrose S2: N3 = N2 + 10%
coconut milk
Campos-Andrada et al. (2001)
A and M a) Cold 72 h + heat 24 h (buds) b) Centrifugation 10 min at 2000 × g then 2 × 5 min at 2000 × g (M)
I: Medium B = KM salts & vitamins + 0.3 M mannitol + 166 mg/l CaCl2
2H2O + 40 mg/l FeEDTA; S: Medium B + 2% sucrose + 0.4% PEG + 2% coconut water + 250–500 mg/l casein hydrolysate
Bayliss et al. (2004)
A Cold or heat not effective I: NN macro- + B5 micro-elements + 0.5 mg/l 2,4-D + 1 mg/l NAA
+ 1 mg/l Kin + 0.5 mg/l BAP + 5% sucrose or maltose + 0.8 g/l L-proline + 0.1 g/l L-serine; S: MS + 0.5 mg/l NAA + 1 mg/l BAP + 0.25 mg/l GA3 + 5%
sucrose or maltose + 0.6% agar
Skrzypek et al. (2008)
Cowpea
A Modified MS + 0.5 mg/l NAA + 0.1 mg/l Kin Ladeinde and Bliss (1977)
I: MS + 1 mg/l NAA + 2 mg/l BAP + 3% sucrose for cvs. Tvu91 and Tvu1987; Cv. Pipo same except for 6% sucrose S: Cv. Tvu 91
using MS + 0.25 mg/l NAA + 0.25 mg/l IAA + 0.5 mg/l 2-iP + 2% sucrose;
Cv. Tvu 1987 MS + 0.05 mg/l BA + 6% sucrose; Cv. Pipo as above but + 0.1 mg/l BAP + 6% sucrose
Mix and Wang (1988)
Continued
M.M. Lulsdorf et al.
Table 11.1. Continued.
Target explantsaStress sequence Mediumb Reference(s)
Mung bean
I: MS + 0.5 mg/l IAA + 1 mg/l Kin; S: MS + 1 mg/l BAP + 0.5 mg/l IBA Arya and Chandra (1989)
A I: MS + 2 mg/l IAA + 2 mg/l 2,4-D + 2 mg/l Kin + 0.7% agar Bajaj and Singh (1980)
Urd bean
A Cold for 72 h (A) I: MS + 2 mg/l 2,4-D + 0.2 mg/l Kin+ 8% sucrose + 70 ml l−1 coconut water Gosal and Bajaj (1988) Pigeon pea
A I: MS + 2 mg/l 2,4-D + 0.2 mg/l Kin + 8% sucrose + 200 mg/lpotato extract + 0.8% agar ; S: MS + 1 mg/l 2,4-D
Gosal and Bajaj (1979)
A MS + 4 mg/l IAA + 2 mg/l Kin Bajaj et al. (1980)
A Cold 5–7 days I: Modified MS + 2 mg/l 2,4-D + 0.2 mg/l Kin; S: as above + 1% agar Sudhakar et al. (1986)
A I: MS + 1.5 mg/l IAA + 0.5 mg/l Kin + 0.8% agar Fougat et al. (1992)
M Cold 3–7 days (buds) I: ½ MS macro + NN micro-elements + vitamins + 0.1 mg/l NAA + 0.1 mg/l BAP + 2% sucrose + 2% glucose; S1: MS + 0.5 mg/l BAP;
S2: MS + 2 mg/l NAA + 0.1 mg/l Kin
Kaur and Bhalla (1998)
A I: B5 + 1.75 mg/lIAA + 2.25 mg/l BAP + 0.22 mg/l Kin + 1.73 mg/l GA3 Narasimham (1999)
A I: MS + 2 mg/l 2,4-D + 0.5 mg/l Kin; S: MS + 2 mg/l BAP Vishukumar et al. (2000)
aA, anthers; M, microspores.
bI, induction; S, subculture; ELS, embryo-like structures; Base media, B5 (Gamborg et al., 1968); HSO (Ochatt et al., 2009); L2 (Phillips and Collins, 1979); ML6 (Kumar et al., 1988);
NLN (White, 1963; Lichter, 1981, 1982); MS (Murashige and Skoog, 1962); RM-IK modified HSO (Ochatt et al., 2009); ML6 (Kumar et al., 1988); R&D (Rao and De, 1987); Enriched B5 (modified B5 by Kao, 1982); B5 ‘long’ (modified B5 by Zhuang et al., 1991; Hu et al., 1996; Carolina Biological Supply Co., Burlington, North Carolina); B5DBIG (modified B5 by Tiwari et al., 2004); Miller’s (Miller, 1963); MSO (de Moraes et al., 2004); modified PTA-15 (Skinner and Liang, 1996); YS amino acids (Yeung and Sussex, 1979); 67 V (Veliky and Martin, 1970); KM (Kao and Michayluk, 1975); NNB5 NN macro-B5 micro-elements (Nitsch and Nitsch, 1969); S&H (Schenk and Hildebrandt, 1972); N6 (Chu, 1978).
Androgenesis and Doubled-haploid Production 165
of anthers in medium RM-IK-17 (modified HSO) (Ochatt et al., 2009) for 10 min fol-lowed by; (iii) electroporation of anthers in the same medium, using 625 V/cm. The final stress treatment was (iv) a 4-day high osmotic medium (563 mmol, RM-IK17/HSO) prior to transfer of anthers onto modified Phillips and Collins (1979) embryo development medium and then maturation medium containing dif-ferent hormones. Plants were regenerated on a modified MS medium with a low amount of BAP (0.10 mg/l) and NAA (0.01 mg mg/l).
Flow cytometry and chromosome counts showed that callus cells were initially haploid but ploidy levels increased with age, resulting in spontaneously doubled haploid embryos and plants.
Lentil (Lens culinaris Medik.
ssp. culinaris)
Lentil (2n = 14) is the least explored spe-cies in terms of haploid technology; calli with a few pro-embryos were obtained but no plants regenerated (Keller and Ferrie, 2002). In another study, buds from cvs CDC Crimson and CDC Robin were cold-treated for 96 h prior to microspore extraction, resulting in multinucleate microspores, but no embryos were regenerated (Croser and Lulsdorf, 2004).
Soybean (Glycine max L. Merr.) Over the past 30 years, there has been an intensive research effort from both the private and public sectors into the cell biology and biotechnology of soybean (2n = 40). However, no routine protocol has been established for haploid or DH plant regeneration, and no DH lines of soybean are currently available (Rodrigues et al., 2004a; Croser et al., 2006).
Initial reports demonstrated induction of callus from anthers (Tang et al., 1973; Ivers et al., 1974; Liu and Zhao, 1986), shoot orga-nogenesis (Yin et al., 1982; Jian et al., 1986) and embryo-like structures (ELS) from anther-derived callus (Zhuang et al., 1991; Hu et al., 1996; Kaltchuk-Santos et al., 1997). In a few
cases, a small number of plants were regen-erated, but the haploid origin of the plants was uncertain (Yin et al., 1982; Jian et al., 1986;
Hu et al., 1996; Zhao et al., 1998; de Moraes et al., 2004; Rodrigues et al., 2004a; Tiwari et al., 2004). A haploid chromosome number (n = 20) was confirmed in a single plant (de Moraes et al., 2004). Detailed cytological stud-ies of soybean anthers were carried out in vivo (Kaltchuk-Santos et al., 1993; da Silva Lauxen et al., 2003) and in vitro (Yin et al., 1982;
Kaltchuk-Santos et al., 1997; Cardoso et al., 2004) describing cellular events related to the androgenic pathway, such as the symmetrical mitotic division of microspores and forma-tion of multinucleate and multicellular pollen grains. Yin et al. (1982) reported multinucle-ate grains after 15–20 days in vitro. Kaltchuk-Santos et al. (1997) were the first to show that these grains were not present at dissection, but started to appear during in vitro incuba-tion, reaching an overall frequency of 0.3% by four weeks of culture.
There is no general consensus regarding the most appropriate microspore develop-mental stage for induction of androgenesis in soybean. Yin et al. (1982) and Ye et al. (1994) found that the early- to mid-uninucleate stage was best for induction. Later reports suggested the mid- to late uninucleate and early binucleate stage of pollen development as appropriate (Kaltchuk-Santos et al., 1997;
da Silva Lauxen et al., 2003; Cardoso et al., 2004). This could be due to the propensity of soybean to have varying developmental stages within the same bud, thereby mak-ing it difficult to establish the original pollen source.
There has been little consensus on the effect of pre-treatment stress on androgen-esis from soybean. To date, authors have focused on testing temperature stress applied to the buds prior to, or directly after, anther or microspore isolation and culture (Liu and Zhao, 1986; Zhuang et al., 1991; Rodrigues et al., 2005b). Hu et al. (1996) recommended the use of sonication to improve sterilization of buds prior to anther isolation. Sonication is now showing potential under testing in our laboratories as an effective elicitation stress in a range of species (Ochatt and Croser, unpub-lished results).
For most species, androgenesis requires an auxin, a cytokinin or a combination of both in the medium (Smýkal, 2000), with soy-bean most likely requiring both (Table 11.1).
In general, B5 medium with 16 organic com-pounds (‘B5 long’) (Zhuang et al., 1991) and with Yeung’s amino acids (Yeung and Sussex, 1979) is appropriate for anther culture. De Moraes et al. (2004) obtained one confirmed haploid plant (2n = 20), following induction of embryogenic calli from anthers on this basal medium supplemented with 2.0 mg/l 2,4-D, 0.5 mg/l BAP, 9% sucrose and 0.25%
phytagel. This result further confirms the finding of Hu et al. (1996) that 2,4-D is essen-tial for soybean microspore callus induction, although Rodrigues et al. (2004b) noted that this growth regulator favours morphogenic response from sporophytic tissue.
Cardoso et al. (2004) showed that a high percentage of soybean microspores doubled their chromosome number within the first ten days of culture, suggesting spontaneous dou-bling may be at a rate high enough to avoid the requirement for an artificial doubling step.
However, it also makes determination of the androgenic origin of regenerated plants more difficult. Rodrigues et al. (2004a) confirmed that soybean androgenic and somatic ELS were induced simultaneously under the same culture conditions. The presence of both het-erozygous and homozygous ELS within the same culture (but not within the same anther) confirmed that somatic embryogenesis and androgenesis were promoted under identi-cal conditions. Zhuang et al. (1991) demon-strated that calli derived from anthers in the first three months of culture were mainly of anther somatic tissue in origin. If this initial callus was removed upon transfer of anthers to fresh medium, four weeks later a few newly grown calli developed embryoids that were more likely of haploid origin. Another strategy to overcome somatic embryogenesis is to culture isolated microspores that are free of the somatic anther tissue. This technique has been applied widely in other species, but rarely in soybean (Liu and Zhao, 1986;
Rodrigues et al., 2006).
While genotypic effects have been recog-nized in soybean, there is little discussion of the effect of donor plant growth conditions,
which can have a profound effect on embryo-genic response. Soybean protocols use anthers collected from the field (Zhuang et al., 1991;
Kaltchuk-Santos et al., 1997; da Silva Lauxen et al., 2003; Cardoso et al., 2004; de Moraes et al., 2004; Rodrigues et al., 2004b) in contrast to most other species, where donor plants are grown under controlled conditions.
Common bean (Phaseolus vulgaris L.)
Given the first report of bean (2n = 22) anther culture (Haddon and Northcote, 1976), little progress has been made in this species in 34 years (Table 11.1). However, the androgenic origin of callus cells could not be determined in the first study because DNA analysis showed only diploid to polyploid chromosome levels.
In contrast, Peters et al. (1977) reported near equal amounts of haploid and diploid cal-lus cells with fewer than 3% of cells showing polyploidy. Tai and Cheng (1990) cultured anthers of common bean on B5 medium with 2 mg/l 2,4-D and 1 mg/l kinetin. Bean cal-lus growth was the poorest among the four legume species tested. Origin of the callus cells is unknown since ploidy levels were not determined. Muñoz and co-workers (Muñoz and Baudoin, 1994, 2001/2002; Muñoz, et al., 1992, 1993) conducted a more detailed study into bean anther culture (Table 11.1). In 1992, these authors reported that the early to mid-uninucleate microspore stage was the most responsive to androgenesis induction and that a larger size of Petri dish (55 mm dia-meter) resulted in more callus growth than smaller ones (35 mm). A few modifications to the MS base medium (Veliky and Martin, 1970) were also tried for better callus growth.
The medium for anther induction was modi-fied to MS macro- and micro-nutrients, B5 vitamins, 2 g/l casein hydrolysate, 2.5%
sucrose and 2 mg/l each of 2,4-D and kinetin (Muñoz and Baudoin, 2001/2002). Cold pre-treatment of anthers did not have a beneficial effect. Callus cells during the early growth stages were predominantly haploid but, with age, ploidy levels increased, thus indicating spontaneous doubling of chromosomes.
Androgenesis and Doubled-haploid Production 167
Lupin (Lupinus spp.)
To date, there has been no confirmed report on haploid embryo or plantlet regeneration from any of the four grain lupin species. Sator (1985) first obtained callus production fol-lowing anther culture of Lupinus luteus and Lupinus angustifolius. Ormerod and Caligari (1994) produced cotyledonary-stage embryos from microspores that were released from cultured anthers of Lupinus albus, but no plants were regenerated. Campos-Andrada et al. (2001) demonstrated in vivo pollen dim-orphism in pearl lupin. Culture of the iso-lated microspores led to symmetrical division and procallus formation. Bayliss et al. (2004) reported isolated microspore-derived pro-embryos in L. albus and L. angustifolius and, most recently, Skryzpek et al. (2008) achieved callus induction from microspores released from anthers of L. albus, L. angustifolius and L. luteus. A feature of these studies was the spontaneous release of microspores into the surrounding medium after anther dehis-cence during culture, similar to that seen in Nicotiana tabacum L. Bayliss et al. (2004) com-pared this natural dehiscence with a mechan-ical microspore isolation system. All reports agree that the uninucleate and/or early binu-cleate microspore stage is optimal in lupin.
Bayliss et al. (2004) obtained haploid pro-embryos from isolated microspores in L. albus and L. angustifolius but found further embryo development to be restricted by the failure of the outer exine layer to rupture. Pro-embryos were induced from microspores that were mechanically isolated from buds stored at 4°C for 72 h and then cultured for 24 h at 32°C (Kao and Michayluk, 1975). The mechanical isola-tion method included a 10 min centrifugaisola-tion step at 2000 × g, more vigorous than that used as a stress treatment for enhancing androgene-sis in chickpea (Grewal et al., 2009). After the 24 h heat and starvation treatment, microspores were transferred to modified KM medium.
This transfer resulted in an osmotic stress treatment, similar in nature to that described for haploid plant production in other legumes by Grewal et al. (2009) and Ochatt et al. (2009).
It appears that the best androgenic response, observed by Bayliss et al. (2004),
came after a rigorous stress treatment of cold, heat, centrifugation, starvation and osmotic stress, thus providing further evidence of the efficacy of combining stress agents for induction of androgenesis from the grain legumes. In contrast, Skrzypek et al. (2008) reported cold and heat pre-treatment either did not improve, or was inhibitory, to callus induction from anthers of L. albus, L. angus-tifolius and L. luteus. This report compared field- with glasshouse-grown donor ma terial, observing that androgenic response was higher in the field-grown plants. The results of Skrzypek et al. (2008) contrasted with those of Bayliss et al. (2004) with regard to the pollen wall limiting further androgenic develop-ment. However, no cytological evidence was presented to support this observation. If the outer exine limits embryo development from microspores, electrostimulation may assist in overcoming this issue as one of its effects is to ‘loosen’ the cell wall (Cole, 1968; Neumann and Rosenheck, 1973).
came after a rigorous stress treatment of cold, heat, centrifugation, starvation and osmotic stress, thus providing further evidence of the efficacy of combining stress agents for induction of androgenesis from the grain legumes. In contrast, Skrzypek et al. (2008) reported cold and heat pre-treatment either did not improve, or was inhibitory, to callus induction from anthers of L. albus, L. angus-tifolius and L. luteus. This report compared field- with glasshouse-grown donor ma terial, observing that androgenic response was higher in the field-grown plants. The results of Skrzypek et al. (2008) contrasted with those of Bayliss et al. (2004) with regard to the pollen wall limiting further androgenic develop-ment. However, no cytological evidence was presented to support this observation. If the outer exine limits embryo development from microspores, electrostimulation may assist in overcoming this issue as one of its effects is to ‘loosen’ the cell wall (Cole, 1968; Neumann and Rosenheck, 1973).