PlantCeU Reports
Plant Cell Reports (1990) 9:443-446
9 Springer-Verlag1990
Plant regeneration through somatic embryogenesis in the forage grass Caucasian bluestem (Bothriochioa caucasica) C. I. Franklin, T. N. Trieu, and R. A. Gonzales Plant Biology Division, The Samuel Roberts Noble Foundation, P.O. Box 2180, Ardmore, OK 73402, USA Received June 18, 1990/Revised version received September 19, 1990 - Communicated by I. K. Vasil
ABSTRACT Plantlets were regenerated from cultured seed explants of the forage grass Caucasianbluestem [Bothriochloa caucasica (Trin.) C.E. Hubbard] via somatic embryogenesis. Embryogenic callus was produced in four weeks when surface sterilized seeds were cultured on a medium containing MSsalts, B-5 vitamins, 12 mM L-proline, 2% sucrose, 0.8% agar and 5p,M 2,4-D. Plantlets were regenerated in 6-8 weeks after culture initiation. Healthy root and shoot systems were produced within three weeks after the plantlets were transferred to a medium lacking 2,4-D. Approximately 95% of the plantlets survived greenhouse acclimation and produced healthy plants and viable seeds. Caucasian bluestem callus cultures exhibit natural resistance to kanamycin. High levels of kanamycin (up to 800 mg/1) did not completely inhibit callus growth. However, the regeneration ofhealthyplantlets was completely inhibited by kanamycin even at low levels (50 rag/l). Key words: Caucasian bluestem, Bothriochloa caucasica, apomixis, somatic embryogenesis. INTRODUCTION Caucasian bluestem, an obligate apomict (Harlan and Chheda 1963), is a high yielding forage grass well adapted to the warm season and different soil conditions in the south central United States (Dalrymple 1978, Sims and Dewald 1982). Seeds of an obligate apomict contain embryos that are produced asexually from the maternal tissue (Nogler 1984). Improvement of an apomictic species through conventional breeding is very difficult, because it cannot be used as a female parent to produce a hybrid. The development of efficient in vitro regeneration protocols may allow the use of biotechnological techniques for the improvement of these crop species. We have developed a tissue culture method to regenerate plants via somatic embryogenesis as an initial step in attempts to improve nutritional qualities and biomass production in Caucasian bluestem. Selection of genetically transformed cells and plantlets is an essential part of DNA-transfer technology. Selection of Offprint requests to: C. I. Franklin
transformants using kanamycin resistance encoded by NPT II gene has been reported only in a few graminaceous species such as Zea mays (Fromm et al. 1986), Triticum monococcum (Lorz et al. 1985) and Oryza sativa (Uchimiya et al. 1986). Many other grasses show natural resistance to kanamycin (Hauptmann et al. 1988). In the present study, we have evaluated the inhibitory effects of kanamycin on Caucasian bluestem callus growth and plantlet production. We also investigated the effect of carbenicillin on Caucasian bluestem callus and plantlets. MATERIALS AND METHODS Preparation of Explants The glumes and other floral tissue (such as stigma) were carefully removed from the seeds of Caucasian bluestem [Bothriochloa caucasica (Trin.) C.E. Hubbard] without damaging the embryo and the endosperm. The seeds were first washed in tap water containing a few drops of commercial liquid detergent. After a thorough rinse in tap water, the seeds were treated with 70% ethanol for 2 min and rinsed again with tap water. The seeds were then soaked in 20% commerical bleach (clorox) solution (plus a few drops of liquid detergent) with constant agitation for 20 min and subsequently rinsed 4 times in sterile distilled water. Callus Culture and Plant Regeneration Surface sterilized seeds were cultured on a medium (referred to as CBS medium) containing MS-salts (Murashige and Skoog 1962), 135vitamins (Gamborg et al. 1968), 12 mM Proline, 2% sucrose, 0.8% agar and 51~M 2,4-dichlorophenoxy acetic acid (2,4-D), under low light conditions (100 lux, fluorescent light) with a 16 hr photoperiod at 23 + 2~ After 4 weeks, callus produced from hypocotyl and epicotyl regions of the germinated seed was selectively removed and transferred to fresh CBS medium and grown under identical light and temperature regimes as described above. Plantlets regenerated from these callus cultures 2-4 weeks after transfer to fresh CBS medium. Callus, along with the plantlets, was sub-cultured on CBS medium at 4 week intervals. Eight to 10 weeks after culture initiation, and subsequently during each sub-culture, plantlets 10 mm or taller were removed
444
Figs. 1-6 1. 2. 3. 4. 5. 6.
Various stages of plant regeneration from seed explants of Caucasian bluestem. Bar indicates l~n. Seeds at the time of culture initiation (after removing glumes and other floral tissue). Callus initiation from germinated seeds, 10 days after culture initiation. Callus produced from the explants 4 weeks after culture initiation. (e = embryogenic callus, ne = non-embryogenic callus) Regeneration of plantlets from the callus 8 weeks after culture initiation. Regenerated plantlets 3 weeks after transfer to 2,4-D free medium, showing root and shoot systems. Regenerated plants grown in the greenhouse for 16 weeks. Note the uniformity in heigh t, flower production and overall appearance.
from the callus and transferred to auxin free m e d i u m (CBS m e d i u m without 2,4-D) contained in Magenta boxes (Magenta Corporation, Chicago, Ill., U.S.A.) and grown under higher light intensity (3500 lux, fluorescent light) with a 16 hr photoperiod at 23 + 2~ Plantlets 3 cm or taller were transferred to soil contained in Magenta boxes, and grown in the greenhouse. Two to 3 days after transfer to the greenhouse, the lids of Magenta boxes were kept partially open. Seven to 10 d a y s later, the plants that survived greenhouse acclimation were transferred to pots.
Effects of 2,4-D concentration The influence of 2,4-D concentration on callus initiation, growth and plantlet production was studied. Nine different 2,4-D concentrations were tested. Ten seed explants per concentration were cultured following the regeneration protocol described above. Nine weeks after culture initiation, data on the number of plantlets, and fresh and d r y weights of callus were collected. Kanamycin dose response Seed explants of Caucasian bluestem were first cul-
445 tured on CBS m e d i u m for 10 days and then transferred to CBS medium containing the antibiotic(s) as described in Table 1. Twenty explants were cultured per treatment, and callus from each explant was cultured in a separate clump. Three weeks after the transfer, fresh and dry weights of callus produced and the number of plantlets regenerated from each explant were determined. Inhibitory effect of kanamycin on callus growth rate was determined using 8-month old callus cultures (subcultured a t 4-week intervals). Callus was cultured on CBS m e d i u m containing the antibiotic(s) as described in Table 2 for a total of 8 weeks. Five replicate callus samples were cultured per treatment. Each sample consisted of one gram (fresh weight) of callus divided into two clumps and cultured in one petridish. After 4 weeks, the fresh weight of callus clumps in each plate was measured and then the callus was transferred to fresh CBS medium containing the same concentrationo f the antibiotic(s).Fresh and dry weights of the calli were measured four weeks after the transfer. In order to simulate identical media formulation that will be used for selection of stable transformants through Agrobacterium mediated transformation, we included carbenicillin(500 rag/l) in all our kanamycin dose response experiments. We also tested ifcarbenicillinshows any inhibitory effect on callus growth, and callus and plantlet production from seed explants b y culturing the callus and seed explants on CBS medium containing 500 mg/1 carbenicillin.
RESULTS A N D DISCUSSION Seeds (Fig 1) germinated 3 days after culture initiation. A translucent and friable non-embryogenic callus first developed from the hypocotyl and the lower portion of the coleoptile by 7-10 days after culture initiation (Fig 2). With continued callus proliferation small pockets of embryogenic callus with the characteristic white color and compact appearance (Vasil, 1988) appeared 4 weeks after culture initiation (Fig 3). Between 6-8 weeks after culture initiation, the embryogenic callus produced several somatic embryos and plantlets with small roots (Fig 4). Both root and shoot Table 1. Effectofkanamycinon callusproductionand plantletregeneration
from Caucasianbluestemseed explants. I
Plant Cell Reports (1990) 9:443-446
9 Springer-Verlag1990
Plant regeneration through somatic embryogenesis in the forage grass Caucasian bluestem (Bothriochioa caucasica) C. I. Franklin, T. N. Trieu, and R. A. Gonzales Plant Biology Division, The Samuel Roberts Noble Foundation, P.O. Box 2180, Ardmore, OK 73402, USA Received June 18, 1990/Revised version received September 19, 1990 - Communicated by I. K. Vasil
ABSTRACT Plantlets were regenerated from cultured seed explants of the forage grass Caucasianbluestem [Bothriochloa caucasica (Trin.) C.E. Hubbard] via somatic embryogenesis. Embryogenic callus was produced in four weeks when surface sterilized seeds were cultured on a medium containing MSsalts, B-5 vitamins, 12 mM L-proline, 2% sucrose, 0.8% agar and 5p,M 2,4-D. Plantlets were regenerated in 6-8 weeks after culture initiation. Healthy root and shoot systems were produced within three weeks after the plantlets were transferred to a medium lacking 2,4-D. Approximately 95% of the plantlets survived greenhouse acclimation and produced healthy plants and viable seeds. Caucasian bluestem callus cultures exhibit natural resistance to kanamycin. High levels of kanamycin (up to 800 mg/1) did not completely inhibit callus growth. However, the regeneration ofhealthyplantlets was completely inhibited by kanamycin even at low levels (50 rag/l). Key words: Caucasian bluestem, Bothriochloa caucasica, apomixis, somatic embryogenesis. INTRODUCTION Caucasian bluestem, an obligate apomict (Harlan and Chheda 1963), is a high yielding forage grass well adapted to the warm season and different soil conditions in the south central United States (Dalrymple 1978, Sims and Dewald 1982). Seeds of an obligate apomict contain embryos that are produced asexually from the maternal tissue (Nogler 1984). Improvement of an apomictic species through conventional breeding is very difficult, because it cannot be used as a female parent to produce a hybrid. The development of efficient in vitro regeneration protocols may allow the use of biotechnological techniques for the improvement of these crop species. We have developed a tissue culture method to regenerate plants via somatic embryogenesis as an initial step in attempts to improve nutritional qualities and biomass production in Caucasian bluestem. Selection of genetically transformed cells and plantlets is an essential part of DNA-transfer technology. Selection of Offprint requests to: C. I. Franklin
transformants using kanamycin resistance encoded by NPT II gene has been reported only in a few graminaceous species such as Zea mays (Fromm et al. 1986), Triticum monococcum (Lorz et al. 1985) and Oryza sativa (Uchimiya et al. 1986). Many other grasses show natural resistance to kanamycin (Hauptmann et al. 1988). In the present study, we have evaluated the inhibitory effects of kanamycin on Caucasian bluestem callus growth and plantlet production. We also investigated the effect of carbenicillin on Caucasian bluestem callus and plantlets. MATERIALS AND METHODS Preparation of Explants The glumes and other floral tissue (such as stigma) were carefully removed from the seeds of Caucasian bluestem [Bothriochloa caucasica (Trin.) C.E. Hubbard] without damaging the embryo and the endosperm. The seeds were first washed in tap water containing a few drops of commercial liquid detergent. After a thorough rinse in tap water, the seeds were treated with 70% ethanol for 2 min and rinsed again with tap water. The seeds were then soaked in 20% commerical bleach (clorox) solution (plus a few drops of liquid detergent) with constant agitation for 20 min and subsequently rinsed 4 times in sterile distilled water. Callus Culture and Plant Regeneration Surface sterilized seeds were cultured on a medium (referred to as CBS medium) containing MS-salts (Murashige and Skoog 1962), 135vitamins (Gamborg et al. 1968), 12 mM Proline, 2% sucrose, 0.8% agar and 51~M 2,4-dichlorophenoxy acetic acid (2,4-D), under low light conditions (100 lux, fluorescent light) with a 16 hr photoperiod at 23 + 2~ After 4 weeks, callus produced from hypocotyl and epicotyl regions of the germinated seed was selectively removed and transferred to fresh CBS medium and grown under identical light and temperature regimes as described above. Plantlets regenerated from these callus cultures 2-4 weeks after transfer to fresh CBS medium. Callus, along with the plantlets, was sub-cultured on CBS medium at 4 week intervals. Eight to 10 weeks after culture initiation, and subsequently during each sub-culture, plantlets 10 mm or taller were removed
444
Figs. 1-6 1. 2. 3. 4. 5. 6.
Various stages of plant regeneration from seed explants of Caucasian bluestem. Bar indicates l~n. Seeds at the time of culture initiation (after removing glumes and other floral tissue). Callus initiation from germinated seeds, 10 days after culture initiation. Callus produced from the explants 4 weeks after culture initiation. (e = embryogenic callus, ne = non-embryogenic callus) Regeneration of plantlets from the callus 8 weeks after culture initiation. Regenerated plantlets 3 weeks after transfer to 2,4-D free medium, showing root and shoot systems. Regenerated plants grown in the greenhouse for 16 weeks. Note the uniformity in heigh t, flower production and overall appearance.
from the callus and transferred to auxin free m e d i u m (CBS m e d i u m without 2,4-D) contained in Magenta boxes (Magenta Corporation, Chicago, Ill., U.S.A.) and grown under higher light intensity (3500 lux, fluorescent light) with a 16 hr photoperiod at 23 + 2~ Plantlets 3 cm or taller were transferred to soil contained in Magenta boxes, and grown in the greenhouse. Two to 3 days after transfer to the greenhouse, the lids of Magenta boxes were kept partially open. Seven to 10 d a y s later, the plants that survived greenhouse acclimation were transferred to pots.
Effects of 2,4-D concentration The influence of 2,4-D concentration on callus initiation, growth and plantlet production was studied. Nine different 2,4-D concentrations were tested. Ten seed explants per concentration were cultured following the regeneration protocol described above. Nine weeks after culture initiation, data on the number of plantlets, and fresh and d r y weights of callus were collected. Kanamycin dose response Seed explants of Caucasian bluestem were first cul-
445 tured on CBS m e d i u m for 10 days and then transferred to CBS medium containing the antibiotic(s) as described in Table 1. Twenty explants were cultured per treatment, and callus from each explant was cultured in a separate clump. Three weeks after the transfer, fresh and dry weights of callus produced and the number of plantlets regenerated from each explant were determined. Inhibitory effect of kanamycin on callus growth rate was determined using 8-month old callus cultures (subcultured a t 4-week intervals). Callus was cultured on CBS m e d i u m containing the antibiotic(s) as described in Table 2 for a total of 8 weeks. Five replicate callus samples were cultured per treatment. Each sample consisted of one gram (fresh weight) of callus divided into two clumps and cultured in one petridish. After 4 weeks, the fresh weight of callus clumps in each plate was measured and then the callus was transferred to fresh CBS medium containing the same concentrationo f the antibiotic(s).Fresh and dry weights of the calli were measured four weeks after the transfer. In order to simulate identical media formulation that will be used for selection of stable transformants through Agrobacterium mediated transformation, we included carbenicillin(500 rag/l) in all our kanamycin dose response experiments. We also tested ifcarbenicillinshows any inhibitory effect on callus growth, and callus and plantlet production from seed explants b y culturing the callus and seed explants on CBS medium containing 500 mg/1 carbenicillin.
RESULTS A N D DISCUSSION Seeds (Fig 1) germinated 3 days after culture initiation. A translucent and friable non-embryogenic callus first developed from the hypocotyl and the lower portion of the coleoptile by 7-10 days after culture initiation (Fig 2). With continued callus proliferation small pockets of embryogenic callus with the characteristic white color and compact appearance (Vasil, 1988) appeared 4 weeks after culture initiation (Fig 3). Between 6-8 weeks after culture initiation, the embryogenic callus produced several somatic embryos and plantlets with small roots (Fig 4). Both root and shoot Table 1. Effectofkanamycinon callusproductionand plantletregeneration
from Caucasianbluestemseed explants. I
