PlantCell Reports
Plant Cell Reports (1992) 11:506- 513
9 Springer-Verlag 1992
Agrobacterium-mediated genetic transformation of oilseed Brassica campestris: Transformation frequency is strongly influenced by the mode of shoot regeneration A. Mukhopadhyay, N. Arumugam, P . B . A . Nandakumar, A.K. Pradhan, V. Gupta, and D. Pental Tata Energy Research Institute, 90 Jor Bagh, New Delhi t t 0 003, India Received M a r c h 19, 1992/Revised version received July 20, 1992 - Communicated by C.F. Quiros
Summary. Protocols were developed for efficient shoot regeneration from hypocotyl and cotyledon explants of oilseed Brassica campestris (brown sarson)cv. 'Pusa Kalyani'. These were used for genetic transformation by an Agrobactetium based binary vector carrying neomycin phosphotransferase (npt) gene and 13-glucuronidase (gus)-intron gene for plant cell specific expression. Transformed plants were recovered from hypocotyl explants at a frequency of 7-13%. Addition of silver nitrate markedly enhanced shoot regeneration in hypocotyl explants under non-selection conditions and was found to be an absolute requirement under selection conditions. Cotyledon explants, inspite of being more regenerative, proved to be highly refractory to transformation. Only two chimeric transformed shoots were obtained from more than 10,000 cotyledons treated withAgrobacterium. In hypocotyl explants, shoot regeneration occurred from the vascular parenchyma both with and without the intervention of callus phase. Only the shoot buds differentiating from callus tissue were positive for GUS activity. In cotyledons, shoot buds originated only directly from the vascular parenchyma, generally at a distance of about 450-625 I.t from the cut surface. Such shoots were negative for GUS activity. Key words: Genetic transformation - Oilseed Brassica campestris -AgrobacteHum - Shoot differentiation
1987, thin cell layers : Charest et al. 1988, hypocotyl : Radke et al. 1988, De Block et al. 1989, microspore embryos : Swanson and Erickson 1989), B. juncea (cotyledon: Mathews et al. 1990, hypocotyl: Barfield and Pua 1991) and B. oleracea (hypocotyl: David and Tempe 1988, De Block et al. 1989). In comparison to the Brassica species that have been transformed, B. campestris (syn. B. rapa) is recalcitrant to in vitro shoot regeneration (Dunwell 1981, Dietert et al. 1982, Murata and Orton 1987, Jain et al. 1988, Narashimhulu and Chopra 1988), the only exception being a report of high frequency regeneration from cotyledonary-petioles (c-petioles) of an oleiferous type of B. campestris (Hachey et al. 1991). In this paper we report the development of transformed plants of oilseed Brassica campestris cv. ?usa Kalyani' using an A. tumefaciens based vector (Vancanneyt et al. 1990) carrying npt gene conferring resistance to kanamycin and gus-intron gene for histochemical identification of transformed tissues and plants. We compare the efficiency of regeneration and transformation of hypocotyl and cotyledon explants and relate the results of genetic transformation to the anatomical studies on the mode of regeneration in the two explant systems.
Materials and methods lh'eparation of erplants. Seeds of Brassica campestris cv. 'Pusa
Introduction
A variety of explants showing morphogenic ability have been used for Agrobacterium mediated transformation of Brassica species. Successfully transformed species and explant sources for each one of these include B. napus (internodes : Fry et al. 1987, stem : Pua et al. Correspondence to: D. Pental
Kalyani' were germinated aseptically on MS (Murashige and Skoog 1962) medium in dark for 2d, then transferred to light (200/tEm-2s "1, 16h photoperiod) and maintained at 25_+1~ Cotyledons with 1-2ram petiole (e-petiole) and hypocotyl segments (0.5 - lcm) were obtained from 4-10d old seedlings. Care was taken to remove the axillary buds present at the cotyledonary axis. Petiolar cut ends were embedded in the medium. Hypocotyl explants were laid lengthwise on the surface of the medium.
Media for shoot regeneration and multiplication. Initially, all
the
507 possible combinations of auxins (NAA. IAA) with cytokinins (BAP, Kn) at concentrations of 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 and l0 mg/l each were tested to establish the suitable combinations for shoot regeneration using 24 c-petiole and 48-50 hypocotyl explants for each treatment. The most responsive of these media for shoot regeneration from hypocotyl and cotyledon explants were tested again along with adjuvants such as silver nitrate (10-200/tM) and coconut water (CW, 10%v/v) which were filter sterilized and added after autoclaving the media. For establishing optimal silver nitrate concentration, at least 50 cotyledon and 120 hypocotyl explants were used for each treatment in each experiment. Explants were scored for percent shoot regeneration after 4-6 wk of culture. Regenerated shoots were multiplied as single node cultures on MS medium with 0.005 mg/l NAA, 0.5 mg/l Kn, 50 mg/1 casein hydrolysate.
outside the right border of T-DNA (see Bevan 1984 and Vancanneyt et al. 1990 for maps of Bin 19 and Bin 19 derivative p35S GUS INT respectively). Hence, lhe number of independent integrations in the plant genome, if any, could be detected by Hind III digests.
Histologicalstudies. Cotyledon (0,2,4,6,8,10,12,14 and 15d after in vitro culture) and hypocotyl explants (0,2,8,16,22 and 28d after in vitro culture)were fixed overnight in ice-cold 6% glutaraldehyde prepared in 0.025M phosphate buffer, pH 6.8. The fixed tissues were passed through methoxyethanol, ethanol, propanol, butanol following Feder and O'Brien (1968) and embedded in glycolmethacrylate. Sections were cut in rotary microtome with glass knife at 5/z thickness, stained with toluidine blue and mounted in DPX mountant.
Agrobacterium mediated transformation. Agrobacterium tumefaciens strain LBA4404 carrying neomycin phosphotransferase (npt) and
Results
O-glucuronidase (gus)-intron genes (Vancanneyt et al. 1990) was grown overnight (220 rpm, 28~ in dark) in 10ml of liquid LB containing 50mg/l rifampicin, 50rag/1 kanamycin and 200 mg/I streptomycin. One ml of this suspension was used to inoculate 100 ml of media of the same composition and allowed to grow overnight. On the third day, the bacteria were pelleted (2100 • g) and resuspended in filter sterilized liquid MSM medium (MS salts and vitamins with 10g/l each of sucrose, glucose and mannitol) supplemented with 30mM MES, 200 ~tM acetosyringone and the three antibiotics mentioned above (pH 5.6). The OD of the bacterial suspension was adjusted to about A600=l.0 prior to culture, subsequently bacteria were grown for 2h, peUeted(2100 :< g) and resuspended in MSM medium. OD was adjusted at A600 = 1.0 and this suspension was used for co-cultivation. Freshly cut explants of cotyledon/hypocotyl and also cotyledons precultured for 1 to 17d on shoot regeneration medium were incubated in the bacterial suspension for lh and co-cultivated on MSM medium supplemented with respective shoot inducing hormones. After 3d the ex~plants were shifted to MS medium supplemented with the same growth hormones, silver nitrate and 200 mg/1 cefotaxime. Selection pressure was applied 4d later by growing the cultures on medium with 20mg/l kanamycin for the cotyledons and 30 mg/l kanamycin for the hypocotyl ex'plants. About 15 cotyledon or 60 hypocotyl explants were cultured per 90mm sterile plastic petridish (Steriware, India). Subculturing was done at 10d intervals. Transformed plants were transferred to soil and grown to maturity following Mukhopadhyay et al. (1991).
Shoot regeneration from hypocot3q explants.
Confirmation of genetic transformation. Cotyledon and hypocotyl explants of non-transformed plants were grown on MS with lmg/l Kn, lmg/l 2,4-D and 5,10,20,30,40,50,80,100 /zg/ml kanamycin. The ex-plants showed slight initial swelling, then turned white and eventually brown on media containing 10 Izg/ml of kanamycin. Hence, leaves from the putative transformants were tested for callusing on MS with lmg/l Kn, lmg/l 2,4-D and 15 /tg/ml kanamycin. Leaves and internodes of the putative transformed shoots were hand-sectioned and used for histochemical localization of GUS activity following Jefferson (1987). Total DNA was isolated from leaves of putative transformants following Dellaporta et al. (19831 and purified on cesium chloride density gradients. DNAs isolated from transformed and control plants were analysed for the presence of npt gene by PCR. Oligos used for npt gene by Hamill et al. (19911 were used with modified cycling parameters of 94~ for 30s, 56~ for 30s and 72~ for 1 min (total 28 cycles). For Southern hybridization, DNAs were digested with Hind III in the presence of 0.01M spermidine. Digested DNAs were run on 0.8% agarose gels (20/zg DNA in each lane) and were further processed following Mukhopadhyay et al. (1991). For npt gene a 0.8 kb Xho I - Xba I fragment from plasmid pRT 100 neo (T6pfer et al. 19891 was used as a probe. Hind III would cut the integrated vector DNA downstream to pA site of npt gene and
Media supplemented with IAA and BAP or Kn induced callusing and rooting responses. Callus formation was generally confined to the lower cut end of the explants. The amount of callus formed increased with increasing concentrations of the hormones. Media containingNAA with Kn also promoted callusing and rooting. However, callus was formed on both the cut ends of the explants. Shoot morphogenesis was observed only on MS containing 3mg/1 NAA and 2mg/1 BAP (SMH medium) at a frequency of less than 1% in explants taken from 5-7d old seedlings. Explants from younger or oldcr seedlings did not show any shoot regeneration. Addition of silver nitrate at different concentrations to SMH medium markedly increased the frequency of shoot differentiation. For 5d old seedling explants, regeneration frequency of around 47% was observed on SMH medium with 10 laM silver nitrate (Fig. 1). Higher concentrations of silver nitrate were required for enhancing the regeneration frequency to around 50% in explants from 6d or 7d old seedlings (Fig. 1). Explants showed callus formation at the cut ends, followed by the differentiation of dark green nodules within 22-25d of culture. These nodules developed into shoot like projections within 10-20d of transfer to SMH medium with 10% v/v CW. Regenerated shoots remained stunted and leafy and showed elongation only after transfer to MS medium with 0.1 mg/1 NAA. The elongated shoots were rooted within 7-10d with 100% frequency on MS supplemented with 1 mg/1 IBA.
Shoot regeneration from cotyledon explants. The best morphogenic response was shown by 5d old explants. As in hypocotyl explants, IAA with BAP or Kn and NAA with Kn generally produced callusing and rooting responses from the cut end of c-petioles. While combinations containing IAA produced nodular callus, the callus on NAA supplemented media was semifriable. Increasing concentrations of hormones induced more callus formation.
508 80
~--~ 5d Hypocotyl
,]: --
o--o 6d Hyloocolyl '~
o--o 5d Colyledon
Table 1. Influence of silver nitrate on shoot regeneration from hypocotyl explants on selection and non-selection media after 8 wk of culture, Explants Silver Percent Percent shoot and media nitrate callusing regeneration (Range) (Mean• (tzM) Control on SMH(non-selection 10 86• 43-49 medium, no
Agrobacterium infection,
Agrobacterium
o--t 20
\ 0
0
50
1O0 150 Silver nitrate)JM
200
Fig. 1. Effect of different concentrations of silver nitrate on shoot regeneration. Data were taken from three independent experiments. Vertical bars indicate standard deviations. Critical difference (CD) at p=0.05 are 3.4,6.6, 3.8 and 6.7 for 5d,6d,7d old hypocotyl and 5d old cotyledon explants, respectively.
Cotyledons showed shoot morphogenesis over a range of NAA and BAP combinations(2-5 mg/l of each). The optimum medium for shoot regeneration was found to be MS medium with 3 mg/l NAA and 5 mg/1 BAP (SMC medium), showing around 40% shoot regeneration after 14-18d of culture. Media promoting callus formation at the petiolar cut ends greatly suppressed the frequency of shoot differentiation. Addition of silver nitrate significantly increased the frequency of shoot regeneration (Fig. 1). Shoots developed on SMC medium and addition of CW was not necessary. Elongation and rooting of the differentiated shoots occurred as described for the hypocotyl explants.
Genetic Transformation of h)pocoO,l explants. All the transformation experiments were done with explants from 5d old seedlings as these showed around 90% survival (observed as lack of browning or necrosis) after co-cultivation with Agrobacterium. Preliminary experiments showed that after 3d co-cultivation on MSM medium, 52% of the explants were positive for GUS activity. Inclusion of silver nitrate did not influence the percentage of cultures showing GUS expression and hence, was omitted from the medium during co-cultivation. Silver nitrate was an absolute requirement for obtaining transformed shoots on selection medium. In three different experiments, when explants (about 1500 in each experiment) were subjected to Agrobacterium infection and plated on regeneration media with six different concentrations of silver nitrate, it was observed
infected, on SMH medium with 30/tg/ml km and 200 #g/ml cefotaxime *CD (p=0.05)
10 30 50 70 90 120
21_+2.51 30-+2.41 33_.1.82 33• 45-+2.49 42-+3.16
0 0 0 1-2 7-13 0
4.98
*CD = Critical Difference, calculated only forAgrobacteh'um infected treatments.
that higher concentrations (7-9 times) of silver nitrate were required for shoot regeneration on selection medium than on non-selection medium (Table 1). Agrobacterium infected explants took 30-40d to form shoot primordia as compared to the normal regeneration period of 25-28 d. Regenerated shoots were grown further and rooted in the presence of 30 ~tg/ml kanamycin. Leaf and petiole tissues from 16 randomly selected putative transformants that were regenerated and rooted in the presence of kanamycin were tested for callusing on kanamycin containing media and for GUS expression. All the plants were found to be positive for both the tests. Total DNAs were isolated from four of these transformants. PCR analysis showed the presence of npt gene in all the transformants (Fig. 2A). Southern hybridization analysis showed the presence of one to three insertions of npt gene in the transformed plants (Fig. 2B). Transformed plants were transferred to soil. Different parts of the transformed plants were tested for GUS activity and were found to be positive.
Genetic transfomtation of co6,ledon explants. More than 10,000 explants were co-cultivated with
Agrobacterium. After 3d of co-cultivation on MSM medium about 47% of the explants showed GUS expression.Agrobactetium treated explants showed about 50% shoot regeneration on non-selection medium in the presence of 90 laM silver nitrate. However, irrespective of the silver nitrate concentration added (30,50,70,90,100,120,150 I.tM), Agrobacterium infected c-petioles rarely produced green shoots on kanamycin containing medium. Generally, the emerging green buds turned white or purple on selection medium within 7d of emergence or after the first subculture.
509
Fig. 2 A-B. Analysis of total genomic DNA for the detection of npt gene in the transgenicplants. A : PCR analysisof kanamycinresistant transgenicplants showing the presence of an expected 0.7kb DNA fragmentfrom the structural region of npt gene. B : Southern analysis for studying the integration pattern of npt gene. DNAs were digested with Hind [II and hybridizedto a 0.8 kb fragment from the structural region of the npt gene. Lane 1, control; lane 2-5, DNA from transgenic plants regeneratedfrom hypocotylexplants;lane 6-7~DNA from chimeric transgenic plants regenerated from cotyledon explants.
9Cotyledon explants, precultured on SMC with 901aM silver nitrate for 2,4,6,8,10,12 and 17d were given fresh cuts and subjected toAgrobacterium infection. None of these cultures produced any green shoots on selection medium. In one experiment 315 explants were allowed to differentiate shoots in the absence of kanamycin after 3d of co-cultivation. A total of 268 shoots (independent events) that regenerated were subcultured on selection medium (MS with 20 I.tg/ml kanamycin) and only two shoots survived. When leaves from these two shoots were tested for callusing on kanamycin (151.tg/ml) containing medium, only a few segments callused suggesting a probable chimeric nature of the shoots. Single node segments from these shoots were micropropagated in an attempt to break-up the chimera. Only one shoot each from plant 1 and 2 rooted on selection medium. Tissues from these putative transformants were used for DNA isolation for PCR and Southern analyses. To test for any Agrobacterium contamination tissues were streaked on YEB medium, but no bacterial growth was detected. The presence of expected 0.7kb npt gene in the total DNA of these shoots was detected by PCR (Fig. 2A) but could not be detected by Southern hybridization (Fig. 2B). The chimeric nature of these shoots could have led to the dilution of the limited number of copies of npt gene in the extracted total DNA. Leaf tissues taken from different nodes of the plants that were transferred to soil were subjected to GUS test. Some of these stained positive for GUS activity. No GUS activity was observed in the floral parts. These observations confirm the chimeric nature of these plants.
Anatomy of shoot bud differentiation and localization of GUS activity in hypocotyl explant. Longitudinal sections of 2-3d old explants showed that the vascular parenchyma (vp) cells present at the cut surface became densely cytoplasmic and started dividing after 48h of culture. The zone of active cell division extended up to a depth of about 625-7501.t posterior to
the cut end (Fig. 3A). Histological study of explants at different time intervals revealed two different modes of shoot differentiation from the vp cells. In mode I cell divisions continued at the cut surface giving rise to an unorganized callus that projected beyond the cut end. Histology of 22d and older cultures revealed de novo formation of nests of thin walled, densely cytoplasmic cells in the matrix of comparatively vacuolated cells in the peripheral regions of the callus tissue (Fig. 3B). 9Vascular connections were gradually established between the mother explant and the meristematic nodules. These regions subsequently differentiated into shoot primordia which were visible at the surface of callus tissue after a growth period of 28-30d. In mode II of differentiation shoot bud primordia developed from the dividing vp cells 450-5001.t posterior to the cut surface in an adventitious manner without an intervening unorganized growth phase (Fig. 3B, arrow II). Such primordia grew through the cortical cells and later emerged through the epidermis. The two modes of differentiation described above were observed both in the non-cocultivated and in the Agrobactenum infected explants. However, the latter showed less callusing and a delay in the onset of shoot primordia differentiation compared to the former. The shoots emerging from the vp cells via mode II were highly suppressed in infected explants and only in a few instances emerged as white or pale green shoots that eventually turned white. In the initial stages of culture, GUS activity was localized in the vp cells close to the cut surface (upto 100 I-t) and occasionally in patches in the cortical region and vp cells posterior to the cut end. In the later stages intense GUS activity was observed in the meristematic shoot primordia that were formed de novo from the callus tissue (Fig. 4A). Shoot buds that emerged directly from the vp cells in an adventitious manner did not show any GUS activity.
Anatomy of shoot bud differentiation and localization of GUS activity in cotyledon explants. As in the hypocotyl explants, divisions were observed in the vp cells after 2d in culture. The zone of active cell
510
Fig. 3 A-D. Histology of shoot bud differentiation from hypocotyl and cotyledon explants - c, callus; sm, shoot meristem; tr, tracheid; vp, vascular parenchyma. A : L.S. of hypocotyl explant from 3d old culture showing proliferation of the vascular parenchyma cells near the cut surface (x 35). B : L.S. of hypocotyl explant from 22d old culture showing differentiation of shoot meristems from the vascular parenchyma posterior to cut surface (mode II, arrow II) and from the periphery of callused vascular parenchyma cells (mode I, arrow I). Black line indicates the cut end of the explant (x 9-5.5). C : L.S. of cotyledon explant from 5d old culture showing proliferation of the vascular parenchyma cells near the cut surface (x 53.5), (Inset : Enlarged view of meristematic cells at the cut surface). I) : L.S. of cotyledon explant from 10el old culture showing endogenous differentiation of shoot meristem from vascular parenchyma cells via mode II (x 34.5).
511
Fig. 4 A-B. Hand sections of Agrobacterium infected hypocotyl and cotyledon explants showing GUS activity, c, callus; mn,meristematic nodule; A : L.S. of hypocotyl explant after 30d of culture showing GUS activity in the meristematic nodules formed on the callus. Note the vascular connections being established with the meristematic nodules (x 18.5). B : l,.S. of cotyledon explant after 18d of culture showing GUS activity only in the cells surrounding the developing shoot meristems that have been marked by an arrow (x 26.5).
division extended to about 375-5001x posterior to the cut end (Fig. 3C). However, there were only limited divisions at the cut end and n o shoot primordia differentiated from this tissue. The mode II of regeneration, described above for the hypocotyl explants, was prominent in the cotyledon explants. The differentiation of shoot primordia generally occurred at a depth of 450-6251~ posterior to the cut end (Fig. 3D). These primordia either developed singly after differentiation or proliferated through the formation of axillary buds in close succession, thereby giving an appearance of differentiation near the cut surface in sections of 28-30d old c-peti01es. In the infected explants only a few cortical cells (up to 1001~ posterior to the cut surface) and vp cells at the cut surface stained positive for GUS activity at early stages of culture. No GUS activity was detected in the shoot primordia that differentiated directly from the vp cells posterior to the cut surface. However, patches of cells in the cortical tissue immediately adjacent to the shoot meristems were positive for GUS activity (Fig. 4B). Discussion
Of the various growth hormone combinations tried for shoot regeneration in B. campestris hypocotyl and cotyledon explants, combinations of NAA and BAP proved superior to any other hormone combinations.
Similar observations have been earlier recorded for shoot morphogenesis in cotyledon explants of B. campestris (Narashimhulu and Chopra 1988, Hachey et al. 1991). It was also observed in the present study that shoot regeneration in hypoeotyl explants was optimal on media containing higher auxin to cytokinin ratio whereas cotyledons required higher cytokinin to auxin ratio. Silver nitrate, an inhibitor of ethylene action, is known to stimulate morphogenesis in many monocot and dicot species (Purnhauser et al. 1987, De Block 1988, Songstad et al. 1988, Chraibi et al. 1991, Marton and Browse 1991) including Brassicas (Biddington et al. 1988, Chi and Pua 1989, De Block et al. 1989, Chi et al. 1990, Pua 1990, Barfield and Pua 1991). In the present study we found that silver nitrate enhanced shoot regeneration from both cotyledon and hypocotyl explants, especially from the latter. It was also observed that higher concentration of silver nitrate (70-90 I.tM) was required to induce shoot morphogenesis under selective culture conditions. The production of ethylene is known to increase under stress conditions (Liberman 1979, Yang and Hoffman 1984), and this could be the reason for the higher concentration of silver nitrate required for shoot morphogenesis in the selection medium. In a preliminary study we observed a 1.4 to 1.7 times increase in the production of ethylene in vitro when
512 Agrobacterium infected hypocotyl cultures were subjected to antibiotic stress of the selection medium as compared to Agrobacterium treated cultures maintained on non-selection medium. The essential role of silver nitrate in obtaining transformed plants ofB. oleracea has been earlier reported by De Block et al. (1989). It is, therefore, important to determine the effective concentration of silver nitrate separately for selection conditions to obtain transformed shoots. In the present study, the hypocotyl explants proved amenable for transformation, whereas it was extremely difficult to transform the cotyledons. Our anatomical studies have revealed two different modes of shoot regeneration from the vascular parenchyma cells of B. campesttis explants. In mode I, vp cells at the cut surface gave rise to callus tissues which subsequently differentiated shoots de novo. In mode II, shoots regenerated directly from the vp cells that were posterior to the cut surface by 450-6251.t. In hypocotyl explants, differentiation occurred by both mode I and mode II. However, transformed shoots could be regenerated only via mode I of shoot differentiation. Similar intermediary callusing phases were described for generating transformed plants from B. napus hypocotyl explants (De Block et al. 1989) andArabidopsis thaliana cotyledonary explants (Schmidt and Willmitzer 1988). However, no anatomical evidence on the mode of regeneration was presented in these cases. In the present study, shoot regeneration occurred from the cotyledon explants only by mode II and only two chimeric transformed shoots were obtained from more than 10,000 explants used for transformation. It is possible that the meristematic cells, competent to regenerate and situated 450-6251~ away from the cut surface, either were not accessible to the bacteria or were not competent physiologically to get transformed. Our extensive attempts to transform precultured cotyledon explants that had preformed meristems via mode II also failed to produce any transgenic plants. Meristem tissues are known to be refractory to transformation or at best yield chimeric transformants at very low frequencies (Feldmann et al. 1987, Schrammeijer et al. 1990, Gould et al. 1991). In Brassicas, while cotyledon explants show high frequency of shoot regeneration (Narashimhulu and Chopra 1988, Chi et al. 1990, Sharma et al. 1990, Hachey et al. 1991) success in genetic transformation using this explant is limited only to B. napus (Moloney et al. 1989). They ascribed their success to the easy accessibility of the bacteria to the morphogenic cells at the cut surface. Probably the process of shoot regeneration in this case followed the mode I pathway described in our study. However, the method of shoot regeneration described by Hachey et al. (1991) for B. campestfis is essentially similar to mode II described in this paper. Based on our observations, we feel that this
protocol would prove refractory to genetic transformation. Perhaps, a medium that induces shoot differentiation by mode I pathway from cotyledonary petioles of B. campestris could give high frequency transformation. This point needs further investigation. The regeneration and transformation systems developed from hypocotyl explants of B. campesttis in this study could be used for introducing useful foreign genes into this crop species. Acknowledgements. Technical assistance was provided by Mr. B.S.Yadav. The research is supported by a CEC grant number ECII0193-IND(BA) and DST grant number SP/SO-D-79/87.
References Barfield DG, Pua EC (1991) Gene transfer in Brassicajuncea using Agrobacterium tumefaciens-mediated transformation. Plant Cell Reports 10:308-314 Bevan M (1984) Binary transformation vectors for plant transform ation. Nucl Acids Res 12:8711-8721 Biddington NL, Sutherland RA, Robinson FIT (1988) Silver nitrate increases embryo production in anther culture of Brussel sprouts. Ann Bot 62:181-185 Charest PJ, Holbrook LA, Gabard J, lyer VN, Miki BL (1988) Agrobacterium-mediated transformation of thin cell layer explants from Brassica napus L. Theor Appl Genet 75:438-445 Chi GL, Pua EC (1989) Ethylene inhibitors enhances de novo shoot regeneration from cotyledons of Brassica campestris ssp. chinensis (chinese cabbage) in vitro. Plant Sci 64:243-250 Chi GL, Barfield DG, Sim GE, Pua EC (1990) Effect of AgNO 3 and aminoethoxyvinylglycine on in vitro shoot and root organogenesis from seedling explants of recalcitrant Brassica genotypes. Plant Cell Reports 9:195-198 Chraibi KM, Latche A, Roustan JP, Fallot J (1991) Stimulation of shoot regeneration from cotyledons of Helianthus annuus by the ethylene inhibitors silver and cobalt. Plant Cell Reports 10:204-207. David C, Tempe J (1988) Genetic transformation of caulifower (Brassica oleracea L. var. Botrytis)by Agrobacterium rhizogenes. Plant Cell Reports 7:88-91 De Block M (1988) Genotype independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens. Theor Appl Genet 76:767-774 De Block M, De Brouwer D, Tenning P (1989) Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the bat" and neo genes in the transgenic plants. Plant Physiol 91:694-701 Dellaporta SL, Wood J, Hicks JB (1983) A plant molecular DNA minipreparation version : II. Plant Mol Biol Reporter 1:19- 21. Dietert MF, Barron SA, Yoder OC (1982) Effects of genotype on ht vitro culture in the genus Brassica. Plant Sci Lett 26:233- 240 Dunwell JM (1981) In vitro regeneration from excised leaf discs of three Brassica species. J Exp Biol 32:789-799 Feder N, O'Brien TP (1968) Plant microtechnique : Some inciples and new methods. Am J Bot 55:123-142 Feldmann KA, Marks MD (1987) Agrobacterittm-mediated transformation of germinating seeds of Arabidopsis thaliana : A non- tissue culture approach. Mol Gen Genet 208:1-9 Fry J, Barnason A, Horsch RB (1987) Transformation of Brassica napus with Agrobacterium tumefaciens based vectors. Plant Cell Reports 6:321-325 Gould J, Devey M, Hasegawa O. Ullian EC, Peterson G, Smith RH (1991) Transformation of Zea mays L. using Agrobacterium ntmefaciens and the shoot apex. Plant Physiol 95:426-434
513 Itachey JE, Sharma KK, Moloney MM (1991) Efficient shoot regeneration of Brassica campestris using cotyledon explants cultured in vitro. Plant Cell Reports 9:549-554 tlamill JD, Rounsley S, Spencer A, Todd G, Rhodes MJC (1991) The use of polymerase chain reaction in plant transformation studies. Plant Cell Reports 10:221-224 Jain RK, Chowdhury JB, Sharma DR, Friedt W (1988) Genotypic and media effects on plant regeneration from cotyledon explant cultures of some Brassica species. Plant Cell Tissue Organ culture 14:197-206 Jefferson RA (1987) Assaying chimeric genes in plant : the GUS gene fusion system. Plant Mol Biol Reporter 5:387-405 Lieberman M (1979) Biosynthesis and action of ethylene. Ann Rev Plant Physiol 30:533-591 Marton L, Browse J (1991) Fascile transformation of Arabidopsis. Plant Cell Reports 10:235-239 Mathews H, Bharathan N, Litz RE, Narayanan KR, Rao PS, Bhatia CR (1990) Transgenic plants of mustard Brassicajuncea (L.) Czern and Coss. Plant Sci 72:245-252 Moloney MM, Walker JM, Sharma KK (1989) High efficiency transformation of Brassica napus usingAgrobacterium vectors. Plant Cell Reports 8:238-242 Mukhopadhyay A, Topfer R, Pradhan AK, Sodhi YS, Steinbiss HH, Schell J, Pental D (1991) Efficient regeneration ofBrassica oleracea hypocotyl protoplasts and high frequency genetic transformation by direct DNA uptake. Plant Cell Reports 10:375-379 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol Plant 15:472- 493 Murata M, Orton T (1987) Callus initiation and regeneration capacities in Brassica species. Plant Cell Tissue Org Cult 11:111-123 Narasimhulu SB, Chopra VL (1988) Species specific shoot regeneration response of cotyledonary explants of Brassicas. Plant Cell Reports 7:104-106 Pua EC, Mehra Palta A, Na~mrF, Chua NH (1987) Transgenic plants of Brassica napus L. Bio/Technolog-y 5:815-817 Pua EC (1990) Somatic embryogenesis and plant regeneration from hypoeotyl protoplasts of Brassicajuncea (L.) Czern and Coss. Plant Sci 68:231-238
Purnhauser L, Medgyesy P, Czake M, Dix PJ, Marton L (t987) Stimulation of shoot regeneration in Triticum aestivum and Nicotiana phtmbaginifolia Viv. tissue culture using the ethylene inhibitor AgNO 3. Plant Cell Reports 6:1-4 Radke SE, Andrews BM, Moloney MM, Crouch ML, Kridl JC, Knauf VC (1988) Transformation of Brassica napus using Agrobacterittm tumefaeiens : developmentally regulated expression of a reintroduced napin gene. Theor Appl Genet 75:685-694 Schmidt R, Willmitzer L (1988) High efficiency Agrobactetium tumefaciens-mediated transformation of Arabidopsis thaliana leaf and cotyledon explants. Plant Cell Reports 7:583-586 Schrammeijer B, Sijmons PC, van den Etzen PJM, Hoekema A (1990) Meristem transformation of sunflower via Agrobacterium. Plant Cell Reports 9:55-60 Sharma KK, Bhojwani SS, Thorpe TA (1990) Factors affecting high frequency differentiation of shoots and roots from cotyledon explants of Brassica juncea (L.) Czern. Plant Sci 66:247-253 Songstad DD, Duncan DR, Wildholm JM (1988) Effect of 1- amin ocyclopropane-l-carboxylic acid, silver nitrate and norbornadiene on plant regeneration from maize callus cultures. Plant Cell Reports 7:262-265 Swanson EB, Erickson LR (1989) Haploid transformation in Brassica napus using an octopine-producing strain of Agn'obacterium tumefaciens. Theor Appl Genet 78:831-835 T6pfer R, Gronenborn B, Schell J, Steinbiss HH (1989) Uptake and transient expression of chimeric gene in seed-derived embryos. The Plant Cell 1:133-139 T6pfer R, Prols M, Schell J, Steinbiss HH (1988) Transient gene expression in tobacco protoplasts : II. Comparison of the reporter gene systems for CAT. NPTII and GUS. Plant Cell Reports 7:225-228 Vancanneyt G, Schmidt R, O'Connor-Sanchez A, Willmitzer L, Rocha-Sosa M (1990) Construction of an intron-containing marker gene : Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium mediated plant transformation. Mol Gen Genet 220:245-250 Yang SF, Hoffman NE (1984) Ethylene biosynthesis and its regulation in higher plants. Ann Rev Plant Physiol 35:155-189
Plant Cell Reports (1992) 11:506- 513
9 Springer-Verlag 1992
Agrobacterium-mediated genetic transformation of oilseed Brassica campestris: Transformation frequency is strongly influenced by the mode of shoot regeneration A. Mukhopadhyay, N. Arumugam, P . B . A . Nandakumar, A.K. Pradhan, V. Gupta, and D. Pental Tata Energy Research Institute, 90 Jor Bagh, New Delhi t t 0 003, India Received M a r c h 19, 1992/Revised version received July 20, 1992 - Communicated by C.F. Quiros
Summary. Protocols were developed for efficient shoot regeneration from hypocotyl and cotyledon explants of oilseed Brassica campestris (brown sarson)cv. 'Pusa Kalyani'. These were used for genetic transformation by an Agrobactetium based binary vector carrying neomycin phosphotransferase (npt) gene and 13-glucuronidase (gus)-intron gene for plant cell specific expression. Transformed plants were recovered from hypocotyl explants at a frequency of 7-13%. Addition of silver nitrate markedly enhanced shoot regeneration in hypocotyl explants under non-selection conditions and was found to be an absolute requirement under selection conditions. Cotyledon explants, inspite of being more regenerative, proved to be highly refractory to transformation. Only two chimeric transformed shoots were obtained from more than 10,000 cotyledons treated withAgrobacterium. In hypocotyl explants, shoot regeneration occurred from the vascular parenchyma both with and without the intervention of callus phase. Only the shoot buds differentiating from callus tissue were positive for GUS activity. In cotyledons, shoot buds originated only directly from the vascular parenchyma, generally at a distance of about 450-625 I.t from the cut surface. Such shoots were negative for GUS activity. Key words: Genetic transformation - Oilseed Brassica campestris -AgrobacteHum - Shoot differentiation
1987, thin cell layers : Charest et al. 1988, hypocotyl : Radke et al. 1988, De Block et al. 1989, microspore embryos : Swanson and Erickson 1989), B. juncea (cotyledon: Mathews et al. 1990, hypocotyl: Barfield and Pua 1991) and B. oleracea (hypocotyl: David and Tempe 1988, De Block et al. 1989). In comparison to the Brassica species that have been transformed, B. campestris (syn. B. rapa) is recalcitrant to in vitro shoot regeneration (Dunwell 1981, Dietert et al. 1982, Murata and Orton 1987, Jain et al. 1988, Narashimhulu and Chopra 1988), the only exception being a report of high frequency regeneration from cotyledonary-petioles (c-petioles) of an oleiferous type of B. campestris (Hachey et al. 1991). In this paper we report the development of transformed plants of oilseed Brassica campestris cv. ?usa Kalyani' using an A. tumefaciens based vector (Vancanneyt et al. 1990) carrying npt gene conferring resistance to kanamycin and gus-intron gene for histochemical identification of transformed tissues and plants. We compare the efficiency of regeneration and transformation of hypocotyl and cotyledon explants and relate the results of genetic transformation to the anatomical studies on the mode of regeneration in the two explant systems.
Materials and methods lh'eparation of erplants. Seeds of Brassica campestris cv. 'Pusa
Introduction
A variety of explants showing morphogenic ability have been used for Agrobacterium mediated transformation of Brassica species. Successfully transformed species and explant sources for each one of these include B. napus (internodes : Fry et al. 1987, stem : Pua et al. Correspondence to: D. Pental
Kalyani' were germinated aseptically on MS (Murashige and Skoog 1962) medium in dark for 2d, then transferred to light (200/tEm-2s "1, 16h photoperiod) and maintained at 25_+1~ Cotyledons with 1-2ram petiole (e-petiole) and hypocotyl segments (0.5 - lcm) were obtained from 4-10d old seedlings. Care was taken to remove the axillary buds present at the cotyledonary axis. Petiolar cut ends were embedded in the medium. Hypocotyl explants were laid lengthwise on the surface of the medium.
Media for shoot regeneration and multiplication. Initially, all
the
507 possible combinations of auxins (NAA. IAA) with cytokinins (BAP, Kn) at concentrations of 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 and l0 mg/l each were tested to establish the suitable combinations for shoot regeneration using 24 c-petiole and 48-50 hypocotyl explants for each treatment. The most responsive of these media for shoot regeneration from hypocotyl and cotyledon explants were tested again along with adjuvants such as silver nitrate (10-200/tM) and coconut water (CW, 10%v/v) which were filter sterilized and added after autoclaving the media. For establishing optimal silver nitrate concentration, at least 50 cotyledon and 120 hypocotyl explants were used for each treatment in each experiment. Explants were scored for percent shoot regeneration after 4-6 wk of culture. Regenerated shoots were multiplied as single node cultures on MS medium with 0.005 mg/l NAA, 0.5 mg/l Kn, 50 mg/1 casein hydrolysate.
outside the right border of T-DNA (see Bevan 1984 and Vancanneyt et al. 1990 for maps of Bin 19 and Bin 19 derivative p35S GUS INT respectively). Hence, lhe number of independent integrations in the plant genome, if any, could be detected by Hind III digests.
Histologicalstudies. Cotyledon (0,2,4,6,8,10,12,14 and 15d after in vitro culture) and hypocotyl explants (0,2,8,16,22 and 28d after in vitro culture)were fixed overnight in ice-cold 6% glutaraldehyde prepared in 0.025M phosphate buffer, pH 6.8. The fixed tissues were passed through methoxyethanol, ethanol, propanol, butanol following Feder and O'Brien (1968) and embedded in glycolmethacrylate. Sections were cut in rotary microtome with glass knife at 5/z thickness, stained with toluidine blue and mounted in DPX mountant.
Agrobacterium mediated transformation. Agrobacterium tumefaciens strain LBA4404 carrying neomycin phosphotransferase (npt) and
Results
O-glucuronidase (gus)-intron genes (Vancanneyt et al. 1990) was grown overnight (220 rpm, 28~ in dark) in 10ml of liquid LB containing 50mg/l rifampicin, 50rag/1 kanamycin and 200 mg/I streptomycin. One ml of this suspension was used to inoculate 100 ml of media of the same composition and allowed to grow overnight. On the third day, the bacteria were pelleted (2100 • g) and resuspended in filter sterilized liquid MSM medium (MS salts and vitamins with 10g/l each of sucrose, glucose and mannitol) supplemented with 30mM MES, 200 ~tM acetosyringone and the three antibiotics mentioned above (pH 5.6). The OD of the bacterial suspension was adjusted to about A600=l.0 prior to culture, subsequently bacteria were grown for 2h, peUeted(2100 :< g) and resuspended in MSM medium. OD was adjusted at A600 = 1.0 and this suspension was used for co-cultivation. Freshly cut explants of cotyledon/hypocotyl and also cotyledons precultured for 1 to 17d on shoot regeneration medium were incubated in the bacterial suspension for lh and co-cultivated on MSM medium supplemented with respective shoot inducing hormones. After 3d the ex~plants were shifted to MS medium supplemented with the same growth hormones, silver nitrate and 200 mg/1 cefotaxime. Selection pressure was applied 4d later by growing the cultures on medium with 20mg/l kanamycin for the cotyledons and 30 mg/l kanamycin for the hypocotyl ex'plants. About 15 cotyledon or 60 hypocotyl explants were cultured per 90mm sterile plastic petridish (Steriware, India). Subculturing was done at 10d intervals. Transformed plants were transferred to soil and grown to maturity following Mukhopadhyay et al. (1991).
Shoot regeneration from hypocot3q explants.
Confirmation of genetic transformation. Cotyledon and hypocotyl explants of non-transformed plants were grown on MS with lmg/l Kn, lmg/l 2,4-D and 5,10,20,30,40,50,80,100 /zg/ml kanamycin. The ex-plants showed slight initial swelling, then turned white and eventually brown on media containing 10 Izg/ml of kanamycin. Hence, leaves from the putative transformants were tested for callusing on MS with lmg/l Kn, lmg/l 2,4-D and 15 /tg/ml kanamycin. Leaves and internodes of the putative transformed shoots were hand-sectioned and used for histochemical localization of GUS activity following Jefferson (1987). Total DNA was isolated from leaves of putative transformants following Dellaporta et al. (19831 and purified on cesium chloride density gradients. DNAs isolated from transformed and control plants were analysed for the presence of npt gene by PCR. Oligos used for npt gene by Hamill et al. (19911 were used with modified cycling parameters of 94~ for 30s, 56~ for 30s and 72~ for 1 min (total 28 cycles). For Southern hybridization, DNAs were digested with Hind III in the presence of 0.01M spermidine. Digested DNAs were run on 0.8% agarose gels (20/zg DNA in each lane) and were further processed following Mukhopadhyay et al. (1991). For npt gene a 0.8 kb Xho I - Xba I fragment from plasmid pRT 100 neo (T6pfer et al. 19891 was used as a probe. Hind III would cut the integrated vector DNA downstream to pA site of npt gene and
Media supplemented with IAA and BAP or Kn induced callusing and rooting responses. Callus formation was generally confined to the lower cut end of the explants. The amount of callus formed increased with increasing concentrations of the hormones. Media containingNAA with Kn also promoted callusing and rooting. However, callus was formed on both the cut ends of the explants. Shoot morphogenesis was observed only on MS containing 3mg/1 NAA and 2mg/1 BAP (SMH medium) at a frequency of less than 1% in explants taken from 5-7d old seedlings. Explants from younger or oldcr seedlings did not show any shoot regeneration. Addition of silver nitrate at different concentrations to SMH medium markedly increased the frequency of shoot differentiation. For 5d old seedling explants, regeneration frequency of around 47% was observed on SMH medium with 10 laM silver nitrate (Fig. 1). Higher concentrations of silver nitrate were required for enhancing the regeneration frequency to around 50% in explants from 6d or 7d old seedlings (Fig. 1). Explants showed callus formation at the cut ends, followed by the differentiation of dark green nodules within 22-25d of culture. These nodules developed into shoot like projections within 10-20d of transfer to SMH medium with 10% v/v CW. Regenerated shoots remained stunted and leafy and showed elongation only after transfer to MS medium with 0.1 mg/1 NAA. The elongated shoots were rooted within 7-10d with 100% frequency on MS supplemented with 1 mg/1 IBA.
Shoot regeneration from cotyledon explants. The best morphogenic response was shown by 5d old explants. As in hypocotyl explants, IAA with BAP or Kn and NAA with Kn generally produced callusing and rooting responses from the cut end of c-petioles. While combinations containing IAA produced nodular callus, the callus on NAA supplemented media was semifriable. Increasing concentrations of hormones induced more callus formation.
508 80
~--~ 5d Hypocotyl
,]: --
o--o 6d Hyloocolyl '~
o--o 5d Colyledon
Table 1. Influence of silver nitrate on shoot regeneration from hypocotyl explants on selection and non-selection media after 8 wk of culture, Explants Silver Percent Percent shoot and media nitrate callusing regeneration (Range) (Mean• (tzM) Control on SMH(non-selection 10 86• 43-49 medium, no
Agrobacterium infection,
Agrobacterium
o--t 20
\ 0
0
50
1O0 150 Silver nitrate)JM
200
Fig. 1. Effect of different concentrations of silver nitrate on shoot regeneration. Data were taken from three independent experiments. Vertical bars indicate standard deviations. Critical difference (CD) at p=0.05 are 3.4,6.6, 3.8 and 6.7 for 5d,6d,7d old hypocotyl and 5d old cotyledon explants, respectively.
Cotyledons showed shoot morphogenesis over a range of NAA and BAP combinations(2-5 mg/l of each). The optimum medium for shoot regeneration was found to be MS medium with 3 mg/l NAA and 5 mg/1 BAP (SMC medium), showing around 40% shoot regeneration after 14-18d of culture. Media promoting callus formation at the petiolar cut ends greatly suppressed the frequency of shoot differentiation. Addition of silver nitrate significantly increased the frequency of shoot regeneration (Fig. 1). Shoots developed on SMC medium and addition of CW was not necessary. Elongation and rooting of the differentiated shoots occurred as described for the hypocotyl explants.
Genetic Transformation of h)pocoO,l explants. All the transformation experiments were done with explants from 5d old seedlings as these showed around 90% survival (observed as lack of browning or necrosis) after co-cultivation with Agrobacterium. Preliminary experiments showed that after 3d co-cultivation on MSM medium, 52% of the explants were positive for GUS activity. Inclusion of silver nitrate did not influence the percentage of cultures showing GUS expression and hence, was omitted from the medium during co-cultivation. Silver nitrate was an absolute requirement for obtaining transformed shoots on selection medium. In three different experiments, when explants (about 1500 in each experiment) were subjected to Agrobacterium infection and plated on regeneration media with six different concentrations of silver nitrate, it was observed
infected, on SMH medium with 30/tg/ml km and 200 #g/ml cefotaxime *CD (p=0.05)
10 30 50 70 90 120
21_+2.51 30-+2.41 33_.1.82 33• 45-+2.49 42-+3.16
0 0 0 1-2 7-13 0
4.98
*CD = Critical Difference, calculated only forAgrobacteh'um infected treatments.
that higher concentrations (7-9 times) of silver nitrate were required for shoot regeneration on selection medium than on non-selection medium (Table 1). Agrobacterium infected explants took 30-40d to form shoot primordia as compared to the normal regeneration period of 25-28 d. Regenerated shoots were grown further and rooted in the presence of 30 ~tg/ml kanamycin. Leaf and petiole tissues from 16 randomly selected putative transformants that were regenerated and rooted in the presence of kanamycin were tested for callusing on kanamycin containing media and for GUS expression. All the plants were found to be positive for both the tests. Total DNAs were isolated from four of these transformants. PCR analysis showed the presence of npt gene in all the transformants (Fig. 2A). Southern hybridization analysis showed the presence of one to three insertions of npt gene in the transformed plants (Fig. 2B). Transformed plants were transferred to soil. Different parts of the transformed plants were tested for GUS activity and were found to be positive.
Genetic transfomtation of co6,ledon explants. More than 10,000 explants were co-cultivated with
Agrobacterium. After 3d of co-cultivation on MSM medium about 47% of the explants showed GUS expression.Agrobactetium treated explants showed about 50% shoot regeneration on non-selection medium in the presence of 90 laM silver nitrate. However, irrespective of the silver nitrate concentration added (30,50,70,90,100,120,150 I.tM), Agrobacterium infected c-petioles rarely produced green shoots on kanamycin containing medium. Generally, the emerging green buds turned white or purple on selection medium within 7d of emergence or after the first subculture.
509
Fig. 2 A-B. Analysis of total genomic DNA for the detection of npt gene in the transgenicplants. A : PCR analysisof kanamycinresistant transgenicplants showing the presence of an expected 0.7kb DNA fragmentfrom the structural region of npt gene. B : Southern analysis for studying the integration pattern of npt gene. DNAs were digested with Hind [II and hybridizedto a 0.8 kb fragment from the structural region of the npt gene. Lane 1, control; lane 2-5, DNA from transgenic plants regeneratedfrom hypocotylexplants;lane 6-7~DNA from chimeric transgenic plants regenerated from cotyledon explants.
9Cotyledon explants, precultured on SMC with 901aM silver nitrate for 2,4,6,8,10,12 and 17d were given fresh cuts and subjected toAgrobacterium infection. None of these cultures produced any green shoots on selection medium. In one experiment 315 explants were allowed to differentiate shoots in the absence of kanamycin after 3d of co-cultivation. A total of 268 shoots (independent events) that regenerated were subcultured on selection medium (MS with 20 I.tg/ml kanamycin) and only two shoots survived. When leaves from these two shoots were tested for callusing on kanamycin (151.tg/ml) containing medium, only a few segments callused suggesting a probable chimeric nature of the shoots. Single node segments from these shoots were micropropagated in an attempt to break-up the chimera. Only one shoot each from plant 1 and 2 rooted on selection medium. Tissues from these putative transformants were used for DNA isolation for PCR and Southern analyses. To test for any Agrobacterium contamination tissues were streaked on YEB medium, but no bacterial growth was detected. The presence of expected 0.7kb npt gene in the total DNA of these shoots was detected by PCR (Fig. 2A) but could not be detected by Southern hybridization (Fig. 2B). The chimeric nature of these shoots could have led to the dilution of the limited number of copies of npt gene in the extracted total DNA. Leaf tissues taken from different nodes of the plants that were transferred to soil were subjected to GUS test. Some of these stained positive for GUS activity. No GUS activity was observed in the floral parts. These observations confirm the chimeric nature of these plants.
Anatomy of shoot bud differentiation and localization of GUS activity in hypocotyl explant. Longitudinal sections of 2-3d old explants showed that the vascular parenchyma (vp) cells present at the cut surface became densely cytoplasmic and started dividing after 48h of culture. The zone of active cell division extended up to a depth of about 625-7501.t posterior to
the cut end (Fig. 3A). Histological study of explants at different time intervals revealed two different modes of shoot differentiation from the vp cells. In mode I cell divisions continued at the cut surface giving rise to an unorganized callus that projected beyond the cut end. Histology of 22d and older cultures revealed de novo formation of nests of thin walled, densely cytoplasmic cells in the matrix of comparatively vacuolated cells in the peripheral regions of the callus tissue (Fig. 3B). 9Vascular connections were gradually established between the mother explant and the meristematic nodules. These regions subsequently differentiated into shoot primordia which were visible at the surface of callus tissue after a growth period of 28-30d. In mode II of differentiation shoot bud primordia developed from the dividing vp cells 450-5001.t posterior to the cut surface in an adventitious manner without an intervening unorganized growth phase (Fig. 3B, arrow II). Such primordia grew through the cortical cells and later emerged through the epidermis. The two modes of differentiation described above were observed both in the non-cocultivated and in the Agrobactenum infected explants. However, the latter showed less callusing and a delay in the onset of shoot primordia differentiation compared to the former. The shoots emerging from the vp cells via mode II were highly suppressed in infected explants and only in a few instances emerged as white or pale green shoots that eventually turned white. In the initial stages of culture, GUS activity was localized in the vp cells close to the cut surface (upto 100 I-t) and occasionally in patches in the cortical region and vp cells posterior to the cut end. In the later stages intense GUS activity was observed in the meristematic shoot primordia that were formed de novo from the callus tissue (Fig. 4A). Shoot buds that emerged directly from the vp cells in an adventitious manner did not show any GUS activity.
Anatomy of shoot bud differentiation and localization of GUS activity in cotyledon explants. As in the hypocotyl explants, divisions were observed in the vp cells after 2d in culture. The zone of active cell
510
Fig. 3 A-D. Histology of shoot bud differentiation from hypocotyl and cotyledon explants - c, callus; sm, shoot meristem; tr, tracheid; vp, vascular parenchyma. A : L.S. of hypocotyl explant from 3d old culture showing proliferation of the vascular parenchyma cells near the cut surface (x 35). B : L.S. of hypocotyl explant from 22d old culture showing differentiation of shoot meristems from the vascular parenchyma posterior to cut surface (mode II, arrow II) and from the periphery of callused vascular parenchyma cells (mode I, arrow I). Black line indicates the cut end of the explant (x 9-5.5). C : L.S. of cotyledon explant from 5d old culture showing proliferation of the vascular parenchyma cells near the cut surface (x 53.5), (Inset : Enlarged view of meristematic cells at the cut surface). I) : L.S. of cotyledon explant from 10el old culture showing endogenous differentiation of shoot meristem from vascular parenchyma cells via mode II (x 34.5).
511
Fig. 4 A-B. Hand sections of Agrobacterium infected hypocotyl and cotyledon explants showing GUS activity, c, callus; mn,meristematic nodule; A : L.S. of hypocotyl explant after 30d of culture showing GUS activity in the meristematic nodules formed on the callus. Note the vascular connections being established with the meristematic nodules (x 18.5). B : l,.S. of cotyledon explant after 18d of culture showing GUS activity only in the cells surrounding the developing shoot meristems that have been marked by an arrow (x 26.5).
division extended to about 375-5001x posterior to the cut end (Fig. 3C). However, there were only limited divisions at the cut end and n o shoot primordia differentiated from this tissue. The mode II of regeneration, described above for the hypocotyl explants, was prominent in the cotyledon explants. The differentiation of shoot primordia generally occurred at a depth of 450-6251~ posterior to the cut end (Fig. 3D). These primordia either developed singly after differentiation or proliferated through the formation of axillary buds in close succession, thereby giving an appearance of differentiation near the cut surface in sections of 28-30d old c-peti01es. In the infected explants only a few cortical cells (up to 1001~ posterior to the cut surface) and vp cells at the cut surface stained positive for GUS activity at early stages of culture. No GUS activity was detected in the shoot primordia that differentiated directly from the vp cells posterior to the cut surface. However, patches of cells in the cortical tissue immediately adjacent to the shoot meristems were positive for GUS activity (Fig. 4B). Discussion
Of the various growth hormone combinations tried for shoot regeneration in B. campestris hypocotyl and cotyledon explants, combinations of NAA and BAP proved superior to any other hormone combinations.
Similar observations have been earlier recorded for shoot morphogenesis in cotyledon explants of B. campestris (Narashimhulu and Chopra 1988, Hachey et al. 1991). It was also observed in the present study that shoot regeneration in hypoeotyl explants was optimal on media containing higher auxin to cytokinin ratio whereas cotyledons required higher cytokinin to auxin ratio. Silver nitrate, an inhibitor of ethylene action, is known to stimulate morphogenesis in many monocot and dicot species (Purnhauser et al. 1987, De Block 1988, Songstad et al. 1988, Chraibi et al. 1991, Marton and Browse 1991) including Brassicas (Biddington et al. 1988, Chi and Pua 1989, De Block et al. 1989, Chi et al. 1990, Pua 1990, Barfield and Pua 1991). In the present study we found that silver nitrate enhanced shoot regeneration from both cotyledon and hypocotyl explants, especially from the latter. It was also observed that higher concentration of silver nitrate (70-90 I.tM) was required to induce shoot morphogenesis under selective culture conditions. The production of ethylene is known to increase under stress conditions (Liberman 1979, Yang and Hoffman 1984), and this could be the reason for the higher concentration of silver nitrate required for shoot morphogenesis in the selection medium. In a preliminary study we observed a 1.4 to 1.7 times increase in the production of ethylene in vitro when
512 Agrobacterium infected hypocotyl cultures were subjected to antibiotic stress of the selection medium as compared to Agrobacterium treated cultures maintained on non-selection medium. The essential role of silver nitrate in obtaining transformed plants ofB. oleracea has been earlier reported by De Block et al. (1989). It is, therefore, important to determine the effective concentration of silver nitrate separately for selection conditions to obtain transformed shoots. In the present study, the hypocotyl explants proved amenable for transformation, whereas it was extremely difficult to transform the cotyledons. Our anatomical studies have revealed two different modes of shoot regeneration from the vascular parenchyma cells of B. campesttis explants. In mode I, vp cells at the cut surface gave rise to callus tissues which subsequently differentiated shoots de novo. In mode II, shoots regenerated directly from the vp cells that were posterior to the cut surface by 450-6251.t. In hypocotyl explants, differentiation occurred by both mode I and mode II. However, transformed shoots could be regenerated only via mode I of shoot differentiation. Similar intermediary callusing phases were described for generating transformed plants from B. napus hypocotyl explants (De Block et al. 1989) andArabidopsis thaliana cotyledonary explants (Schmidt and Willmitzer 1988). However, no anatomical evidence on the mode of regeneration was presented in these cases. In the present study, shoot regeneration occurred from the cotyledon explants only by mode II and only two chimeric transformed shoots were obtained from more than 10,000 explants used for transformation. It is possible that the meristematic cells, competent to regenerate and situated 450-6251~ away from the cut surface, either were not accessible to the bacteria or were not competent physiologically to get transformed. Our extensive attempts to transform precultured cotyledon explants that had preformed meristems via mode II also failed to produce any transgenic plants. Meristem tissues are known to be refractory to transformation or at best yield chimeric transformants at very low frequencies (Feldmann et al. 1987, Schrammeijer et al. 1990, Gould et al. 1991). In Brassicas, while cotyledon explants show high frequency of shoot regeneration (Narashimhulu and Chopra 1988, Chi et al. 1990, Sharma et al. 1990, Hachey et al. 1991) success in genetic transformation using this explant is limited only to B. napus (Moloney et al. 1989). They ascribed their success to the easy accessibility of the bacteria to the morphogenic cells at the cut surface. Probably the process of shoot regeneration in this case followed the mode I pathway described in our study. However, the method of shoot regeneration described by Hachey et al. (1991) for B. campestfis is essentially similar to mode II described in this paper. Based on our observations, we feel that this
protocol would prove refractory to genetic transformation. Perhaps, a medium that induces shoot differentiation by mode I pathway from cotyledonary petioles of B. campestris could give high frequency transformation. This point needs further investigation. The regeneration and transformation systems developed from hypocotyl explants of B. campesttis in this study could be used for introducing useful foreign genes into this crop species. Acknowledgements. Technical assistance was provided by Mr. B.S.Yadav. The research is supported by a CEC grant number ECII0193-IND(BA) and DST grant number SP/SO-D-79/87.
References Barfield DG, Pua EC (1991) Gene transfer in Brassicajuncea using Agrobacterium tumefaciens-mediated transformation. Plant Cell Reports 10:308-314 Bevan M (1984) Binary transformation vectors for plant transform ation. Nucl Acids Res 12:8711-8721 Biddington NL, Sutherland RA, Robinson FIT (1988) Silver nitrate increases embryo production in anther culture of Brussel sprouts. Ann Bot 62:181-185 Charest PJ, Holbrook LA, Gabard J, lyer VN, Miki BL (1988) Agrobacterium-mediated transformation of thin cell layer explants from Brassica napus L. Theor Appl Genet 75:438-445 Chi GL, Pua EC (1989) Ethylene inhibitors enhances de novo shoot regeneration from cotyledons of Brassica campestris ssp. chinensis (chinese cabbage) in vitro. Plant Sci 64:243-250 Chi GL, Barfield DG, Sim GE, Pua EC (1990) Effect of AgNO 3 and aminoethoxyvinylglycine on in vitro shoot and root organogenesis from seedling explants of recalcitrant Brassica genotypes. Plant Cell Reports 9:195-198 Chraibi KM, Latche A, Roustan JP, Fallot J (1991) Stimulation of shoot regeneration from cotyledons of Helianthus annuus by the ethylene inhibitors silver and cobalt. Plant Cell Reports 10:204-207. David C, Tempe J (1988) Genetic transformation of caulifower (Brassica oleracea L. var. Botrytis)by Agrobacterium rhizogenes. Plant Cell Reports 7:88-91 De Block M (1988) Genotype independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens. Theor Appl Genet 76:767-774 De Block M, De Brouwer D, Tenning P (1989) Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the bat" and neo genes in the transgenic plants. Plant Physiol 91:694-701 Dellaporta SL, Wood J, Hicks JB (1983) A plant molecular DNA minipreparation version : II. Plant Mol Biol Reporter 1:19- 21. Dietert MF, Barron SA, Yoder OC (1982) Effects of genotype on ht vitro culture in the genus Brassica. Plant Sci Lett 26:233- 240 Dunwell JM (1981) In vitro regeneration from excised leaf discs of three Brassica species. J Exp Biol 32:789-799 Feder N, O'Brien TP (1968) Plant microtechnique : Some inciples and new methods. Am J Bot 55:123-142 Feldmann KA, Marks MD (1987) Agrobacterittm-mediated transformation of germinating seeds of Arabidopsis thaliana : A non- tissue culture approach. Mol Gen Genet 208:1-9 Fry J, Barnason A, Horsch RB (1987) Transformation of Brassica napus with Agrobacterium tumefaciens based vectors. Plant Cell Reports 6:321-325 Gould J, Devey M, Hasegawa O. Ullian EC, Peterson G, Smith RH (1991) Transformation of Zea mays L. using Agrobacterium ntmefaciens and the shoot apex. Plant Physiol 95:426-434
513 Itachey JE, Sharma KK, Moloney MM (1991) Efficient shoot regeneration of Brassica campestris using cotyledon explants cultured in vitro. Plant Cell Reports 9:549-554 tlamill JD, Rounsley S, Spencer A, Todd G, Rhodes MJC (1991) The use of polymerase chain reaction in plant transformation studies. Plant Cell Reports 10:221-224 Jain RK, Chowdhury JB, Sharma DR, Friedt W (1988) Genotypic and media effects on plant regeneration from cotyledon explant cultures of some Brassica species. Plant Cell Tissue Organ culture 14:197-206 Jefferson RA (1987) Assaying chimeric genes in plant : the GUS gene fusion system. Plant Mol Biol Reporter 5:387-405 Lieberman M (1979) Biosynthesis and action of ethylene. Ann Rev Plant Physiol 30:533-591 Marton L, Browse J (1991) Fascile transformation of Arabidopsis. Plant Cell Reports 10:235-239 Mathews H, Bharathan N, Litz RE, Narayanan KR, Rao PS, Bhatia CR (1990) Transgenic plants of mustard Brassicajuncea (L.) Czern and Coss. Plant Sci 72:245-252 Moloney MM, Walker JM, Sharma KK (1989) High efficiency transformation of Brassica napus usingAgrobacterium vectors. Plant Cell Reports 8:238-242 Mukhopadhyay A, Topfer R, Pradhan AK, Sodhi YS, Steinbiss HH, Schell J, Pental D (1991) Efficient regeneration ofBrassica oleracea hypocotyl protoplasts and high frequency genetic transformation by direct DNA uptake. Plant Cell Reports 10:375-379 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol Plant 15:472- 493 Murata M, Orton T (1987) Callus initiation and regeneration capacities in Brassica species. Plant Cell Tissue Org Cult 11:111-123 Narasimhulu SB, Chopra VL (1988) Species specific shoot regeneration response of cotyledonary explants of Brassicas. Plant Cell Reports 7:104-106 Pua EC, Mehra Palta A, Na~mrF, Chua NH (1987) Transgenic plants of Brassica napus L. Bio/Technolog-y 5:815-817 Pua EC (1990) Somatic embryogenesis and plant regeneration from hypoeotyl protoplasts of Brassicajuncea (L.) Czern and Coss. Plant Sci 68:231-238
Purnhauser L, Medgyesy P, Czake M, Dix PJ, Marton L (t987) Stimulation of shoot regeneration in Triticum aestivum and Nicotiana phtmbaginifolia Viv. tissue culture using the ethylene inhibitor AgNO 3. Plant Cell Reports 6:1-4 Radke SE, Andrews BM, Moloney MM, Crouch ML, Kridl JC, Knauf VC (1988) Transformation of Brassica napus using Agrobacterittm tumefaeiens : developmentally regulated expression of a reintroduced napin gene. Theor Appl Genet 75:685-694 Schmidt R, Willmitzer L (1988) High efficiency Agrobactetium tumefaciens-mediated transformation of Arabidopsis thaliana leaf and cotyledon explants. Plant Cell Reports 7:583-586 Schrammeijer B, Sijmons PC, van den Etzen PJM, Hoekema A (1990) Meristem transformation of sunflower via Agrobacterium. Plant Cell Reports 9:55-60 Sharma KK, Bhojwani SS, Thorpe TA (1990) Factors affecting high frequency differentiation of shoots and roots from cotyledon explants of Brassica juncea (L.) Czern. Plant Sci 66:247-253 Songstad DD, Duncan DR, Wildholm JM (1988) Effect of 1- amin ocyclopropane-l-carboxylic acid, silver nitrate and norbornadiene on plant regeneration from maize callus cultures. Plant Cell Reports 7:262-265 Swanson EB, Erickson LR (1989) Haploid transformation in Brassica napus using an octopine-producing strain of Agn'obacterium tumefaciens. Theor Appl Genet 78:831-835 T6pfer R, Gronenborn B, Schell J, Steinbiss HH (1989) Uptake and transient expression of chimeric gene in seed-derived embryos. The Plant Cell 1:133-139 T6pfer R, Prols M, Schell J, Steinbiss HH (1988) Transient gene expression in tobacco protoplasts : II. Comparison of the reporter gene systems for CAT. NPTII and GUS. Plant Cell Reports 7:225-228 Vancanneyt G, Schmidt R, O'Connor-Sanchez A, Willmitzer L, Rocha-Sosa M (1990) Construction of an intron-containing marker gene : Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium mediated plant transformation. Mol Gen Genet 220:245-250 Yang SF, Hoffman NE (1984) Ethylene biosynthesis and its regulation in higher plants. Ann Rev Plant Physiol 35:155-189
