unconventional methods, somatic hybridization via protoplast fusion and interploid sexual hybridization, has become an integral part of mandarin cultivar improvement programs targeting seedlessness worldwide, because they can easily overcome some mentioned
Interspecific hybrids between Diospyros glandulosa (2n = 2x = 30) and D. kaki cv. Jiro (2n = 6x = 90) were produced by electrofusion of protoplasts. Protoplasts were isolated from calli derived from leaf primordia, fused electrically, and cultured by agarose-bead culture using modified KM8p medium. Relative nuclear DNA contents of calli derived from fusion-treated protoplasts were determined by flow cytometry. One-hundred-forty-nine of 166 calli obtained had the nuclear DNA content of the sum of those of D. glandulosa and D. kaki cv. Jiro. RAPD analysis showed that the 149 callus lines yielded specific bands for both D. glandulosa and D. kaki cv. Jiro and they appeared to be interspecific somatic hybrid calli. Shoots were regenerated from 63 of the 149 interspecific hybrid calli. PCR-RFLP of chloroplast DNA analysis, flow cytometric determination of nuclear DNA content, and RAPD analysis revealed that the 63 interspecific hybrid shoot lines contained nuclear genome from both the parents but only chloroplast genome from D. glandulosa. Microscopic observation of root tip cells confirmed that somatic chromosome numbers of the interspecific hybrids were 2n = 8x = 120.
Abstract
Most horticultural plant breeders probably are skeptical of the “potential for crop improvement by genetic manipulation through the use of protoplasts,” which has been professed optimistically in review papers and at genetic engineering conferences and meetings (45). This attitude may be warranted to some degree (43). Certainly, the ability to manipulate quantitatively inherited traits will be very difficult to achieve. Exogenous DNA may be introduced into protoplasts, but the subsequent integration, phenotypic expression, and sexual transmission of the new DNA are prerequisite to any possible exploitation by plant breeders. Significant advances have been and are being made presently, however, toward the realization of somatic cell genetic manipulations by the use of protoplasts. These efforts should increase genetic diversity of available germplasm and provide more efficient means for handling specific traits at various steps in plant breeding.
Petiole protoplasts of the sweetpotato [Ipomoea batatas (L.) Lam.] cultivars Red Jewel and Georgia Jet formed cell walls within 24 hours and divided in 2 to 3 days. Pretreating enzyme solutions with activated charcoal increased the viability and division frequency of protoplasts. Culture of protoplast-donor plants in a medium containing STS did not affect plant growth, protoplasm yield, or viability, but did increase the division frequency. Culture of protoplasts for 24 hours in a medium containing DB, a cell wall synthesis inhibitor, or staining of protoplasts with FDA did not significantly affect division frequency. The division frequency of protoplasts cultured in liquid medium was significantly higher than that of protoplasts cultured in agarose-solidified medium. Cell cycle analysis of petioles and freshly isolated protoplasts showed that the latter has a significantly higher proportion of nuclei in G1 phase. Protoplasts did not initiate DNA synthesis or mitosis within the first 24 hours of culture. Low-frequency regeneration of shoots from protoplast-derived callus was accomplished on MS medium containing 1.0 mg ldnetin/liter when preceded by MS medium modified to contain only (in mg·liter-1) 800 NH4NO3, 1400 KNO3, 0.5 2,4-D, 0.5 kinetin, and 1.0 ABA. Roots produced from protoplast-derived callus formed adventitious shoots after 4 weeks on MS medium containing 2% sucrose, 0.02 mg kinetin/liter and 0.2% Gelrite. Secondary shoot formation from regenerated roots will be a more effective means of obtaining plants from protoplasts than direct shoot regeneration from callus. Chemical names used: silver thiosulfate (STS): 2.6-dichlorobenzonitrile (DB); fluorescein diacetate (FDA): 2.4-diacetate (FDA); 2.4 dichlorophenoxyacetic acid (2,4-D); abscisic acid (ABA).
The genetic improvement of citrus is a challenge because of long generation times, polyembryony, and sexual incompatibilities ( Grosser and Gmitter, 1990 ). To overcome these barriers, protoplast fusion and plant regeneration techniques have gained
Somatic hybridization in citrus using the embryogenic suspension protoplast plus leaf protoplast fusion model can be considered a reliable technique, because hundreds of allotetraploid somatic hybrids have been produced for both scion and
combined with green fluorescent protein of pX-DG-vector ( Chen et al., 2009 ) to yield a fusion protein GFP-SbPIP1. Next, the fusion expression vector pX-DG-SbPIP1 was introduced into onion epidermal cells using a gene gun (PDS-1000; Bio-Rad, Hercules, CA
Protoplast isolation and culture protocols were developed for leaf tissue from 6 kenaf cultivars [Everglades 41 (E41), E71, Guatemala 4 (G4), G45, G51, and Tainung 1]. For protoplast isolation, the best combination of hydrolytic enzymes was cellulysin (1% w/v; Calbiochem) plus macerase (0.5% w/v; Calbiochem), with a 24 hour digestion at 30°C in the dark. Yields reached 7.2 (10)6 protoplasts/g leaf tissue. Protoplast viabilities ranged from 65% to 96%. Minor cultivar differences were observed related to protoplast yield, but all viability estimates were in an acceptable range. Greatest cell division frequencies and plating efficiencies were obtained when protoplasts were initially cultured in liquid medium at a density of 1.0 (10)5 protoplasts/ml. Electrofusion protocols were developed for kenaf protoplasts testing the range from 1200 to 3000 V/cm. A fusion voltage of 2000 V/cm yielded the highest fusion frequency and retained viability above 80%.
Cauliflower protoplasts with male fertile and Ogura male sterile cytoplasm were fused. Organelle reassortment and recombinant mitochondria were found in calli and plants regenerated from the fused protoplasts. Pretreatment (gamma-irradiation or iodoacetate) and protoplasm source (leaf or hypocotyl tissue) were manipulated in a series of fusions to determine their effects on organelle segregation. Some regenerated plants appear to combine Ogura male sterile mitochondria with normal Brassica chloroplasts. Plants with this organelle combination should be free of the cold temperature chlorosis due to incompatibility between the Brassica nucleus and the radish chloroplasts of the Ogura cytoplasm. These plants may have potential for improved cauliflower hybrid production.