The zygotic embryos were recognized in the egg sac and excised out and grown to maturity


It is clearly a marked advantage to use a root stock variety that will reproduce the parent type as nearly true to type as possible by such propagation. Since the embryos developed by cross-fecundated egg cells are likely to be variable, owing to the commingling of genes of different parents, it is evident that the larger number of nucellar embryos developed, the greater will be the proportion of seedlings having the same characters as the mother plant. Citrus nurserymen are interested in the trueness to type of the root stock cultivars—not the zygotes which may be only of interest to the geneticist. Different Citrus species, varieties and hybrids, as pointed out by Webber , Frost , Toxopeus , Torres , Ueno, Iwamasa, and Nishiura , Frost and Soost Cameron and Frost , and others, show considerable difference in the number of nucellar embryos developed. For one who is deeply interested in the subject, all references should be checked. The best summary of the subject is that of Frost and Soost in Table 4-2. Of great concern to the nurserymen, however, are such discrepancies as the Citrus Research Center , Riverside, reporting 54 per cent nucellars for Rough lemon, whereas Torres reports 98 per cent, and a 43 per cent rate for Red lime at CRC, whereas everyone anticipates its nucellar rate as being in the 90 per cent range. The author spoke to Dr. Cameron about this problem, and he assured me he felt that such instances were due to the low number of seedlings examined at CRC for those varieties. Parlevliet and Cameron postulated that one principal dominant gene controls the occurrence of polyembryony,vertical farming companies and that a recessive gene is present in homozygous condition in monoembryonic individuals. Studies by Iwamasa, Ueno and Nishiura generally agree with those of Parlevliet and Cameron, although some geneticists may question this.

Furusato , on polyembryonic studies in Citrus, found with three cultivars that the mean embryo number varied with position on the tree, fruits on the north side of the tree having a higher embryo count than those on the south side of the tree. Mean embryo count was also higher in fruits from old trees than on young trees. The embryo count also tended to be higher in a year of abundant fruit yield than in a year of poor yield. Cameron and Garber pointed out that identical twin hybrids of Citrus x Poncirus arise from strictly sexual seed parents and that identical twins can commonly appear in monoembryonic types. Bitters, et al. demonstrated that multiple embryos can be obtained from monoembryonic types by excising the nucellus tissue from the young ovule and growing the explant on a nutrient media under sterile conditions. In this experiment, trifoliate orange was used as the marker pollen. All of these showed thetrifoliate leaf character and fruit character. However, all the plants derived from the nucellus proved to be different and not as good as the parent plant when grown in the field to fruiting. Not only were lack of the seedlings within the cultivar different, but none of them displayed any trifoliate orange characteristics. The changes had to be due to somatic variation, as the seedlings were not zygotes. Esen and Soost have shown that a high percentage of triploids commonly occur in polyembryonic diploid types as a result of the doubling of the chromosome number of the female gamete. Nishiura, Iwamasa and Ueno report on a monoembryonic trifoliate orange. They indicate that the recessive genes causing monoembryony are present in heterozygous condition in the trifoliate orange. When the flowers from such heterozygous parent trees are self-pollinated or cross-pollinated, monoembryonic hybrids, homozygotes with recessive monoembryonic genes, can arise to some extent through gene recombination, together with polyembryonic hybrids and nucellar seedlings. They conclude that the monoembryonic trees were of zygotic origin. Nakatani, Ikeda, and Kobayashi indicate that a higher percentage of hybrid seedlings may be obtained from a highly polyembryonic variety when grown under high temperature conditions. The total number of embryos per seed are reduced and the percentage of zygotes increases.

Ohta and Furusato found heteroploid seedlings to be a frequent phenomenon in citrus. Most of these plants were 2 N + 1, some were 2 N + 2, and only one 2 N + 3. These seedlings were weak and probably would be discarded in culling from the seed bed to the nursery row. They also found some mixoploid tissue in the root tips. In 1948, I made a large root stock planting at Baldwin Park, California, to ascertain root stock reaction to tristeza . The planting consisted of six tree plots with four replications and Valencia orange scions. Alternate trees were inoculated with tristeza, the others served as checks. The root stocks included a number of trifoliate orange selections, and in particular, a triploid trifoliate. Very poor tree performance was obtained on the tetraploid trifoliate orange which could not be attributed to the tristeza inoculations, or any other factor. A few years later, Dr. F. E. Gardner, then Director of the USDA-ARS-Horticultural Station at Orlando, Florida, also made some plantings on this same source of tetraploid trifoliate. These plantings were in the absence of tristeza and a few years after planting Dr. Gardner reported to me obtaining very poor unexplainable performance on this root stock. In the early 1960’s when I first visited Japan, Dr. Masao Iwamasa also informed me that a tetraploid trifoliate also gave unsatisfactory performance in Japanese trials with Satsuma. There is no logical explanation as to why a tetraploid root stock should not perform well with a diploid scion based on cytological or anatomical compatibility. Perhaps Furusato provides the most plausible answer. Tetraploid trifoliate plants usually have leaves of a darker green color, thicker, and more leathery, larger stomata, and perhaps most important, the main root was thicker and the number of lateral roots was considerably smaller than in the diploids. In a number of citrus groves Furusato found stunted, poorly developed, slow growing trees; this poor performance could not be attributed to other factors. The poor trees were shorter in height, had smaller branches, the trunk diameter was smaller,outdoor vertical plant stands and the number of fruits per tree was markedly reduced. On the tetraploid stocks the scions had smaller leaves, which curled inward and were of a lighter green color. The smaller fruits were firmer in texture than those on diploid stocks.

Furusato states that tetraploid seedlings occur so frequently in Poncirus trifoliata that they cause serious loss to the groves’ owners. The tendency to produce tetraploid seedlings is not just limited to the trifoliate orange, and perhaps neither is the character of the tetraploid root system. The nature of a tetraploid root system could have far-reaching implications. Recently, there has been great interest in somatic hybridization. This technique is based on fusing two distinct protoplasts to create an asexual hybrid which may be impossible to obtain by conventional methods because of incompatibility of the two parents. If the fusion occurs between two diploid protoplasts, then the resultant somatic hybrid is a tetraploid. Recently, according to Bender , Dr. Jude Grosser of the University of Florida’s Lake Alfred Station has succeeded in developing somatic hybrids of Severinia buxifolia and Citrus sinensis. The hybrids are tetraploids with 36 chromosomes. Thirty-year-old grapefruit trees of Severinia, at the Citrus Research Center, Riverside, had the weakest root systems I ever saw. How they sustained the scion physiological, or structurally, to keep the trees from blowing over in a wind, I’ll never know. Thus with an inherent poor root system which may be further reduced because of the tetraploid nature, Dr. Grosser may find it essential, or desirable, to grow haploid plants of both parents so that the haploid protoplasts can be fused to produce the normal diploid hybrid. The important point to make is that here is a method that is successful in producing hybrids which heretofore have been impossible to create. The facts are, however, that there are different phenotypes of Severinia; that Washington navel and Valencia orange as well as Eureka and Lisbon lemons are incompatible on Severinia; that oranges on Severinia are susceptible to tristeza. These are problems which may be overcome. Such hybrid root stocks will not be proven and gain grower acceptance overnight; it may take decades more of research. Esan showed that the chalazal end of the monoembryonic nucellus has an inhibitor present which prevents nucellar embryo formation in monoembryonic citrus types. When the chalazal end of a monoembryonic ovule is “grafted” onto the apical end of the nucellus of a polyembryonic type, the rate of formation of nucellar embryos is reduced. This inhibitor ought to be extracted and identified, as the compound could be useful in breeding experiments with certain species. The number of embryos produced is thus not always an indication that a variety is nucellar or that all multiple embryos are alike. In those varieties producing few nucellar embryos, such as the lemon varieties Eureka and Lisbon, most of the embryos and resultant seedlings would normally be developed from fecundated egg cells. However, those producing a large number of embryos, such as the Rough lemon, would have only a few seedlings developing from fertilized egg cells because in most instances the zygotic embryo aborts, perhaps from inability to compete with the nucellar embryos for nutrients, or maybe there is an inhibitor present.

Recently, Torres, Soost, and Mau-Lastovicka have reported on the use of citrus isozymes as a means of distinguishing nucellars from zygotic seedlings. The use of the isozymes and the practice of excising the zygotic embryo from out of the young ovule before the zygote aborts should aid in producing and identifying a higher percentage of zygotes without the long delay of planting them in the field and waiting for fruiting for more positive examination. The author has felt that for too long the citrus root stock breeder has sacrificed merit for convenience. In other words, root stock crosses have been made knowing a higher percentage of hybrids would be obtained with the parents used, irrespective of the horticultural and pathological benefits of the two parents involved. For more detailed information on nucellar embryony, readers are advised to consult Cameron and Frost and Frost and Soost . Nucellar embryony is not peculiar to citrus, but for root stock purposes most other tree crops do not possess and utilize this useful advantage. Some apples such as Malus sikkimensis, and most mangos, Mangifera indica, have this characteristic, but so far no commercial advantage has been made of this attribute. In the reproduction of certain first-generation hybrids from highly nucellar types, such as the Sampson tangelo and many of the citranges, it has been found that the seedlings in the second generation come as true to type of the first-generation seedlings as if they had been grown from cuttings. In one experiment, 37 seedlings of the Sampson tangelo, grown at the Citrus Experiment Station, Riverside, from fruits taken from a tree open to free cross-pollination, exhibited, at the age of 12 years when in full fruit, almost no recognizable variation. This is an important point to stress. Seedling population of many highly nucellar cultivars may show slight deviations in characters, although they may all be typically Washington navels, Valencia oranges, or Troyer citranges. These slight differences will be in vigor, slightly larger or smaller fruit sizes, earlier maturity, fruit quality, etc. Perhaps these differences may not be due to the fact that they are hybrids, but there might be some slight somatic changes which cause these minute differences. Likewise, progenies of other root stock cultivars, consisting of several hundred seedlings each from Rough lemon, Palestine sweet lime, and the Savage, Cunningham, Rusk, Morton and Coleman citranges up to three years of age, were found to be similarly uniform. Seedlings of the Sanford citrange, however, exhibited great variation and nucellar embryony is apparently less frequent or wanting. Seedlings of the Sanford citrange, in addition, show a high percentage of bark scaling when they got older, similar to psorosis. Seedlings of the Poorman’s orange also exhibit a bark scaling.