水稻花粉培养文献英文3

Vegetal,
de Investigation
Fact&ad de Farmacia, Universidad de Valencia. Avda. Blasco Ibafiez, 13, Valencia-10, Spain y Desarrollo Agrario de Levante, I.N.I.A., Moncada (Valencia), Spain Received 24 November 1980
INTRODUCTION
Since GUHA & MAHESHWARI (1964, 1966) achieved the development of embryos from microspores of Datura innoxia, this technique was succesful with Nicotiuna (BOURGIN & NITSCH, 1967; SUNDERLAND & WICKS, 1969) Lycopersicon esculentum (GRESSHOFF & DOY, 1972), Hyosciamus niger (CORDUAN, 1975), Sohznum tuberosum (DUNWELL & SUNDERLAND, 1973) Brassica oleracea (KAMEYA & HINATA, 1970), Hordeum vulgare (CLAPHAM, 1973), Secale cereale (WENZEL & THOMAS, 1974) Triticum aestivum (WANG et al., 1973) and Zea muys (Institute of genetics, Peking, 1975). Results obtained with other species were less satisfactory. In rice, Oryza sativa L., callus proliferation from anther culture and subsequent organogenesis are generally scarce. The factors affecting callus formation and plant regeneration from in vitro cultured anthers of rice were studied by NIIZEKI & OONO (1968, 1971); HARN (1969); NISHI & MITSUOKA (1969); IYER & RAINA (1972); WANG et al. (1974); LIN et al. (1974); CHEN (1976, 1977) and Hsu & CHEN (1977). Important differences in the behavior of a number of cultivars were found (GUHAMUKHERJEE, 1973); in many of them no pollen grain proliferation was achieved. Haploid plant production from pollen grains cultured in vitro opens a promising field in the experimental mutagenesis as it allows the use of haploid cells or protoplasts for induction and selection of recessive mutations. Anther culture may show proliferation of diploid somatic tissues. To obviate this inconvenience, culture of isolated pollen was attempted. This is also advantageous in 541
INDEX WORDS
Oryza
sativa,
rice, anther
culture,
isolated
pollen
grain.
Factors favouring callus proliferation and subsequent regeneration of plants from pollen grains of rice anthers (Oryza sativa L., cvs. Bahia, Girona, Balilla x Sollana and Sequial) were determined. Cultivar differences in response were found, such as a high rate of haploid plant regeneration. ’ In addition, isolated pollen grain culture was used to induce tissue proliferation outside the anther walls. The frequency ofcallus formation from isolated pollen grains was very low. It was necessary to preculture the anthers before pollen grain separation, in order to accomplish a successful development later. Root differentiation was observed in some of the obtained callus.
M. J. CORNEIA-MARTIN
AND
E. PRIMO-MILL0
improving the efficiency of mutagenic treatments, which is rather low when the target cells are protected by surrounding tissue. The culture of isolated microspores in D. innoxia (NITSCH & NORREEL, 1974) and N. tabacum (NITSCH, 1974) has been approached successfully. Within the gramineae family, culture of isolated pollen has been accomplished in Scale cereale. In the present paper, factors favouring the haploid plant! production through anther culture in four rice cultivars are reported. Isolated pollen grain culture has been used to induce tissue proliferation outside the anther walls.
MATERIALSAND METHODS
Anther culture. Rice plants (cvs. Bahia, Girona, Balilla x Sollana and Sequal) were grown to maturity. Flower panicles were excised, some of them kept at 2-4°C for 48 hours and the others used without cold pre-treatment. The panicles were surface sterilized for 10 minutes in a filtered solution of calcium hypochlorite at 7 % (w/v) and 0.1 % (v/v) Tween 20, followed by three times rinsing in sterile distilled water. Before starting with the cultures some anthers of each spike were squashed in acetocarmine to assessthe stage of pollen grain development. Anthers were placed on a nutrient medium containing MS mineral salts (MURASHIGE & SKOOG, 1962), vitamins according to the method of NITSCH & NITSCH (1965), 3 % (w/v) sucrose, (0.2-2 mg/l) 2.4-dichlorophenoxy acetic acid (2.4-D) or (0.2-2 mg/l) naphtalene acetic acid (NAA) and 0.7% (w/v) agar (Difco Bacto); pH = 5.6. The medium was autoclaved at 120°C for 20 minutes and then distributed in 10 cm plastic Petri dishes. The cultures were placed at 27°C in two different conditions, in the dark and in a 16hour light period (2000 lux of illuminance). Chromosome counts were made on root-tips, stained in Feulgen reagent after hydrolysis in 1 N HCl at 60°C for 11 minutes and squashed in 1% acetic orcein. Isolatedpollen grain culture. Cultivar Balilla x Sollana was used because of its high capacity to form calluses and subsequent regeneration of haploid plants. Before pollen grains were separated, anthers were kept at a temperature of 224°C for 48 hours, and then sterilized and cultured during 3, 7 or 20 days in a medium supplemented with NAA (2 mg/l). At the end of this inductive period, about 90 anthers were placed in a glass homogenizer containing 20 ml of the cultured medium without growth regulators. By pushing the piston slightly, pollen grains were freed and residues of somatic tissue eliminated through a stainless steel mesh having a pore diameter of 0.100 mm. The obtained pollen suspension was centrifuged at low speed (1000 rpm for 5 min.). The pellet was washed with culture medium, precipitated again by centrifuging and finally resuspended in a appropriate amount of medium supplemented with NAA (2 mg/l) and casein hydrolysate (500 mg/l), so that the density was about 104- 10’ pollen grains per ml. The final suspension was poured into Petri dishes (4-6 ml per dish) and incubated in darkness at 28°C. 542