In vitro mutation breeding for salinity tolerance in Citrus

Full text: Mutation breeding began in the 1970's, concentrating on early ripening and high quality spontaneous mutants, subsequently on the induction of seedless mutants by gamma radiation and other mutagens. Extremely early cultivars 'Guoqing 1 to 5' have been obtained from bud mutations in satsuma mandarin and are grown commercially. Other desirable mutants have been found in progenies from gamma ray treated budwood. Recently we introduced plant tissue and cell culture techniques hoping to avoid chimerism and diplontic selection and to obtain solid mutants in a shorter time, resistant to salt, herbicides, low temperature and diseases. I. Methods for cell and protoplast culture. Ovules from fruitlets 1-6 weeks after flowering, cultured in MT supplemented with IAA 0.1 mg/I and KT 1.0 mg/l, gradually produce callus from nucelli. After several passages, habituated callus lines are obtained, which grow very well in MT medium without any growth substances. These callus lines, regardless of the species and cultivars are white, friable, composed of cell clumps of various size and highly embryogenic. When transferred to MT medium containing 2% glycerol and caseinhydrolysat 400 mg/l, they become green and form numerous green globular embryoids within one month. Most of the embryoids derive from single cells. The embryoids could develop roots and shoots forming large numbers of plantlets in a fresh medium. Habituated callus lines have been established in 5 cultivars. Their high embryogenic capacity of single cell origin provides good material for mutation induction. For obtaining protoplasts, the habituated calluses were transferred into liquid MT medium and cultured for two passages on a shaker, then they were macerated with enzyme solution containing 0.3%-0.5% pectinase, 0.3%-0.5% cellulase, MT macroelements and 0.7 M mannitol, at 5.7 pH. After incubation at 25 deg. C for 12-16 hrs., a large number of viable protoplasts could be collected. They were resuspended to a density of 80,000-100,000 protoplasts/ml and kept in liquid MT containing 0.14 M sucrose and 0.46 M mannitol. After 4 days, a few protoplasts resumed division, 2 weeks later, about 15% of the protoplasts have divided once to three times, looking like proembryos. After 2 months, they formed visible globular embryoids, which would develop plantlets after transfer. For plant proliferation, the appropriate media were MT supplemented with BR (Brassinolide) 0.001 mg/ml - 0.02 mg/l and 6BA 3 mg/l - 5 mg/l or 6BA 1 mg/l - 5 mg/l and NAA 0.25 mg/l - 1.0 mg/l. With the combinations of BR and 6BA, even root segments could develop adventitious buds. II. Mutagenic treatment. The mutagens used include gamma radiation, EMS, SA and PYM (Penyanmycin). PYM, a kind of antibiotic, was discovered by the Genetics Research Institute of the Chinese Academy of Science. It is a stronger mutagen than EMS and can induce a large amount of chromosome aberrations. But application of gamma radiation to cells and protoplasts seems to operate more easily, since chemical mutagens usually give rise to breaks in protoplast membrane and loss of protoplasts. The LD50 of gamma radiation, EMS, PYM and SA for Citrus callus cells were 5-7 Krad, 0.3% (25 deg. C, 12-24 hrs.), 0.5-2.5 μg/ml (25 deg. C, 8 hrs.) and 0.01 M - 0.05 M (25 deg. C, 8 hrs.), respectively. Protoplasts were much more sensitive. Decrease of cell and protoplast viability, delay in cell wall regeneration of protoplasts and the first division of cells derived from protoplasts, and reduction in colony formation were effects of mutagenic treatments. Gamma radiation of 7 krad and EMS at 0.2%-0.5% produced 6.50% and 5.98% chromosome aberrations, respectively, but SA very few. Among the aberrations were lagging chromosomes, fragments, bridges, unequal divisions and micronucleates, some of which could lead to sterility and seedless fruit mutants. III. Selection for salt tolerance. The habituated callus cells, having recovered after treatment with mutagens, were exposed to NaCl. Through continuous selection for 5-7 passages, mutant cell lines able to stand 0.8% NaCl were obtained in 4 rootstock varieties and 4 scion cultivars. They still possess embryogenic capacity and have regenerated many plantlets. Growth curve analysis indicated that these NaCI tolerant cell lines, after 3 passages in non-selective medium, kept similar growth increment and cell viability when placed again in NaCI medium. Under salt stress, the soluble protein content decreased significantly in the original cell lines but not in the mutant cell lines. The accumulation of proline in the mutant lines was 30% - 70% higher. Peroxidase activity of the mutant lines was lower and had an additional band compared with the original forms. Also Na+ and Cl- absorption of the mutant lines was different indicating that the mutant lines could avoid NaCI. At present, protoplasts isolated from these mutant cell lines are being cultured to obtain salt tolerant plants from protoplasts and embryoids have already been formed. Next we intend to fuse protoplasts of salt tolerant and non-tolerant lines for complementation test to further clarify the genetic nature of salt-tolerance and to use it perhaps as a genetic marker. Salt-tolerant scion and rootstock plants will be grafted together and checked for their performance under such circumstances. (author)