Medium for induction of callus
Upland cotton cultivar ‘Coker 201’ was used to select the efficiency of callus induction medium. The explants began to swell at both ends of hypocotyls after cultured on MSB medium (Table 1) 5~7 days, the hypocotyls elongated and intumesced. A mass of callus produced 30~35 days. Induction capacity varied in medium (CIM01- CIM12) with different combinations and concentration of hormones. The colors of the callus diverged from fawn to green or yellow green, etc., and the textures were compact, loose, friable and hardy. The induction frequency of callus was higher in the medium supplemented with high concentration of 2, 4-D (0.904 µM) and higher ratio of 2, 4-D/KT concentration (Table 1), indicated that 2, 4-D was an important hormone for the induction of callus.
IBA or IAA was employed to meliorate the callus state reported before (Wang et al. 2006). Callus could be improved to friable and loose texture in light yellow or green yellow color when the IAA (0.571 µM) or IBA (2.460 µM) were added to the medium containing 2, 4-D and KT, but higher frequency of callus induction was obtained using IAA than that of using IBA (Table 1). The ratio of auxin: cytokinin should not be too high in order to reduce the formation of rhixoid.
Callus with loose and friable texture in yellow and light yellow color were easier to differentiate, this result was coincident with previous report (Han et al. 2009). The CIM06, CIM medium supplied with 2, 4-D (0.904 µM), IAA (0.571 µM) and KT (0.465 µM) (Table 1), generated high frequency callus with desirable color and texture, was confirmed to be the most suitable one for callus induction.
Callus induction among different genotypes of Upland cotton
Explants from fifteen Upland cotton cultivars were used to induce callus in the selected medium CIM06, containing MS + 2, 4-D (0.904 µM) + IAA (0.571 µM) + KT (0.465 µM). No obvious difference was observed among these varieties in terms of the time of the callus initiation, color and texture of infant callus in the process of callus induction. The induction rate of loose callus varied from 40.22% to 100.00% (data not shown). The induction rate of 13 cultivars was as higher than 82%, which confirmed further that the medium CIM06 was suitable for callus induction, except induction rate of C1013 and Yumian No.1 were 40.22% and 48.84%, respectively.
Proliferation rates of callus of different genotypes
Loose callus was subcultured every 30 days on the medium supplemented with 5.16 mM NH4NO3, 1.504 M KNO3, 1.48 µM IBA and 0.465 µM KT for proliferation and development. The wet weights of developing callus were recorded each 30 days to calculate the proliferation rate (g/d) of callus.
The fifteen genotypes could be classified as four types based on the proliferation rates of callus (Fig. 1). Category I: Callus proliferated very slowly in dark gray color and soft and wet texture, such as Yumian No. 1, C1006 and C1013. Category II: Callus proliferated in general rate, which was in light yellow color, soft and wet texture. More than half of the genotypes, such as CAU102, were classified in the group. Category III: Callus of Luwu 401, Jiwu 2031, ND 58 and C1013 proliferated fast, and the callus is light yellow in friable and dry texture. Category IV: Callus of C1028 proliferated in a "crazy" mode with firm and hard texture in green color.
Embryogenic callus induction and somatic embryo formation
Callus of different genotypes were subcultured on medium with MSB1 (1.480 µM IBA, 0.465 µM KT), 6.843 mM glutamine and 3.784 mM asparagine to induce embryogenic callus. After one month subcultured on this medium, various callus differentiations were found among the 15 genotypes. The color, texture, number of callus proliferation and embryogenesis callus varied (Table 2), which can be classified into three types (Fig. 2A-C). Category I: 4 varieties, Coker 201, ND 58, Jiwu 2031 and CAU 102 could differentiate into typical embryogenic callus in light yellow or gray color with friable or alveolate structure; category II: 9 varieties formed callus in brown or green color with the compact surface, but were hard to differentiate into embryogenic callus on this medium; category III: callus from C1028 proliferated in a "crazy" mode in dark green color with loose texture, and could not differentiate into embryogenic callus either. Embryogenic callus from the cultivar which had the ability of redifferentiation could produce somatic embryos after two to three times of subculture on this medium.
Development of somatic embryos and plant regeneration
Embryos formed from embryogenesis were transferred onto medium supplemented with seven combinations and concentrations of hormones plus 10.31 mM NH4NO3, 6.843 mM glutamine, and 3.784 mM asparagine for embryo development. Somatic embryos successfully developed into plants after subculturing on this medium for four to five weeks, but the growth and development of the embryos were obviously different (Fig. 3). The germination rate of the embryos with normal root on the medium without hormones was 41%, indicating it was the best among the seven media used in embryo germination. 0.985 µM IBA and 0.465 µM KT (MSB4) could facilitate embryo germination and maturation. Medium containing high concentrations of hormones (MSB2) could increase the rate of abnormal plantlets. The ratio of somatic embryo dedifferentiation was 22.3%, 34.5% and 28.9% when the embryos germinated on medium supplied with MSB4, MSB5 and MSB6, respectively, which indicated that 2, 4-D could result in somatic embryo dedifferentiation and should not be used during embryo germination. The treatment MSB6 with reduced IBA concentration with 2, 4-D (0.492 μM IBA + 0.465 μM KT + 0.452 μM 2, 4-D) showed reduction in somatic embryo dedifferentiation (28.9%) compared to that of MSB5 (34.5%), which indicated that the relatively lower concentrate of IBA would more proper for embryo development and germination.
Somatic embryos also developed from globular embryo, heart shaped embryo, torpedo shaped embryo, cotyledonary shaped embryo to plantlets (Fig. 2D) as previous report (Sun et al. 2009), and the development process was similar to that of zygotic embryo. After the regenerated plantlets grew four to six leaves and normal roots in the flask, all of the rooted plantlets were successfully transplanted to soil pots in green house. All plants recovered, flowered and bore seed after two to three months in green house, the regenerated plantlets had normal fertility (Fig. 2E-K). And these results also indicated that SE in Upland cotton would be more useful with low frequency of chimeras and higher proportion of regenerates.
The solid-liquid alternating culture increased inducing efficiency of embryogenetic callus and somatic embryos
The explants of ‘Coker 201’, ‘Jiwu 2031’ , ‘CAU 102’ and ‘ND 58’ began to swell at both ends of hypocotyls after cultured on CIM (MS medium supplemented with 0.571 µM IAA, 0.465 µM KT and 0.452 µM 2, 4-D), and a mass of callus produced within 25~30 days (Fig. 4A, the abbreviation for Fig. 4A (a), 4B (a) and 4C (a), similar showed below). After subculture of the initial callus on the same medium for 14 to 28 days, the pre-embryogenetic callus was highly variable in color and texture. Typical colors of non-embryogenetic callus were green, light yellow, white, gray, and brown. The types of callus texture include loose, friable and compact.
For embryogenetic callus initiation and maintenance, pre-embryogenetic callus with loose or friable texture and in gray or light yellow (Fig. 4(b)) were chosen to suspension in MS liquid medium and shake for five minutes to disperse the callus. After removing the large particles with 30-mesh stainless steel sieves, the pre-embryogenetic callus was re-suspended in EIML (Fig. 6) and cultured for 14 days until the callus with identical color and texture formed. EIML contained halved NH4NO3, doubled KNO3, and supplied with 2.46 µM IBA+0.698 µM KT, not similar to the hormone-free medium used in establishing and maintaining embryogenic suspension cultures of cotton cultivar ‘Coker 312’ (Trolinder and Goodin, 1987). The callus in identical/similar light yellow and loose texture were chosen for further suspension culture in 50 mL fresh EIML (Fig. 4(c)). After 14~28 days’ inoculation, the embryonic callusor embryogenetic tissues were found in suspension cultures. In this study, the induction frequency of embryogenesis callus by suspension culture was 72.14%, 52.86% and 61.62% in ‘Coker 201’, ‘Jiwu 2031’ and ‘ND 58’, respectively (Table 3), while that in solid culture for Coker 201 was 63.5% (Wu et al. 2004). The induction time to form embryonic callus was shorted from 60~90 days (Wu et al. 2004) to 35~42 days.
For somatic embryo development/maturation, once the embryogenesis callus with similar/identical status of friable texture and yellow color were formed in suspension culture system, embryogenesis callus were filtered with 50-mesh stainless steel sieves, and subcultured onto EMMS (with halved NH4NO3, doubled KNO3, 2.460 µM IBA, 0.698 µM KT, 6.843 mM glutamine and 3.784 mM asparaginate) (Fig. 4(d)). Within 28 days, large amounts embryos were obtained (Fig. 4(e)). At the initial stage of embryos, the percentage of global-embryos counts for 67.5% which indicated that the highly synchronous of embryos development. The embryos in identical status (global embryos) were selected to subculture on the solid medium EGM for germination (Fig. 4(f)). Following placement of the mature embryos on EGM, hypocotyl and root elongation was first observed in 3~7 days (Fig. 4(g)). The percentages of embryos germination were 47.71%, 39.83% and 46.67% in ‘Coker 201’, ‘Jiwu 2031’ and ‘ND 58’, respectively (Table 3). Within 14 days, the majority of the embryos had undergone obvious elongation, and then the cotyledons developed, and plantlets regenerated with at least 2 euphylla in an additional month (Fig. 4(g)).