--F. Locatelli, M. Bertolini and E. Lupotto
Genetic transformation of maize has been recently obtained by the use of the biolistic approach applied to embryogenic regenerable suspension (Gordon-Kamm et al., Plant Cell 2:603-618, 1990). In other cereals, PEG-mediated transformations applied to regenerable protoplast systems have led to transformed regenerated plants. Also in this case, however, the establishment of embryogenic suspension cultures was the keypoint for the successful application of transformation techniques. To date, maize can be regenerated from several genotypes, but optimized friable embryogenic cultures are derived only from a few elite genotypes; among these A188 and its cross with B73 are the most widely used. In addition, other inbreds such as B79 (Lupotto, E and Lusardi, Maydica 33:163-177, 1988), may give origin to friable embryogenic calli, but in these cases suspension cultures have not been established.
In view of an efficient application of transformation techniques to maize cells, the identification of genotypes particularly suitable for tissue culture in giving friable cultures, suspension cultures, and regenerable protoplast systems assumes particular relevance. Furthermore, cultures initiated from agronomically suitable genotypes will facilitate breeding procedures once a new genetic trait is introduced in regenerable cells by genetic transformation.
Since the study on B79 cultures, we have been interested in establishing optimized embryogenic cultures in various maize genotypes, such as inbreds produced at the Section in Bergamo and characterized by outstanding agronomical traits.
A recent series of Lo inbred lines has been released by our Institution; each of these lines is characterized by specific traits including tolerance to pests, diseases and viruses, early vigor, and strong root apparatus (Table 1). Among the lines chosen for this investigation, six (Lo876, Lo904, Lo950, Lo951, Lo964, and Lo1054) are related to the Iowa Stiff Stalk breeding group (SSS), and four (Lo881, Lo924, Lo1056, and Lo1059) are related to the Lancaster or other breeding groups (Bertolini et al., Maydica 36:87-106, 1991). These lines were evaluated per se and in crosses with A188 and B79 for their responsiveness in tissue culture. The aim of the work was to establish embryogenic cultures from each genotype, to test their regeneration capability, and to assay their responsiveness as suspension cultures.
Table 1. Lo inbred lines and their characteristics.
|Lo inbred lines||Some major agronomical characteristics||In vitro responsec|
|Lo876a||stalk rot resistance, stay-green, resistance to BYDV, MDMV, and MRDV||A|
|Lo881b||high yield, stay-green, long-ear, tolerance to plant density||A|
|Lo904a||high yield, low-ear insertion, early vigor||A|
|Lo924b||high yield, stalk quality, early vigor||A|
|Lo950a||high yield, stay-green, stalk quality, tolerance to MDMV||B|
|Lo951a||high yield, stay-green, stalk quality, tolerance to MDMV||B|
|Lo964a||high yield, stay-green, low-ear insertion, stalk quality, tolerance to MDMV||A|
|Lo1054a||high yield, stay-green, stalk quality, strong root system||B|
|*Lo1056b||high yield, stay-green, stalk quality, strong root system||B|
|*Lo1059b||high yield, stay-green, stalk quality, strong root system||B|
Callus cultures were induced from the scutellum of immature embryos on N6I medium (Lupotto and Lusardi, Maydica 33:163-177, 1988) in the presence of 2mg/l 2,4-D, propagated in very dim light (200 lux) conditions on the same medium in the presence of 1mg/l 2,4-D, and regenerated on Murashige and Skoog (MS) medium devoid of hormones in the light (3000 lux). With regard to the callus induction frequency (CIF) the genotypes were grouped in four classes of in vitro responsiveness: A = very low, CIF <10% of the explanted embryos; B = low, CIF 10-20%; C = medium, CIF 20-50%; and D = high, CIF >50%. As indicated in Table 1, Lo inbreds were considered non-responding genotypes in vitro. Callus induction frequency did not exceed the second class of responsiveness (A and B) when compared to inbreds highly responding in vitro, such as A188 and B79. A second parameter evaluated at the beginning of the third subculture after embryo explant was the embryogenic callus induction (ECI), determined by scoring under dissecting microscope for the presence/absence of sectors bearing somatic embryos at the surface. ECI also includes four classes of responsiveness ranging in the same values of percentage as CIF. No embryogenic, or occasionally very few stabilized callus cultures, were developed from Lo inbreds when selfed embryos were explanted. This holds true from greenhouse and field grown donor plants in two sets of experiments. Conversely, crosses of some Lo inbreds with A188 and B79 gave interesting results regarding callus induction, somatic embryogenesis, plant regeneration, and suspension culture establishment. As summarized in Table 2, crosses of various Lo to A188 and B79 inbreds, and their reciprocals, allowed inclusion of almost each genotype in the classes C and D, thus drastically enhancing their in vitro culturability. The five Los showing some callus induction per se, grouped in class B for CIF, improved up to the highest responding class D, in crosses with A188 or with B79. In all cases friable em-bryogenic calli were obtained, more typically type II calli in the crosses with A188. The five less responsive Lo inbreds (grouped
Table 2. In vitro responsiveness of crosses between Lo inbreds and A188
|Class of responsiveness from crosses|
|Class of responsiveness of Lo selfed||LoxA188||A188xLo||LoxB79||B79xLo|
in class A) did improve consistently their performance, except in one case. The inbred Lo904 responded very poorly when used as female parent in the cross Lo904 x A188, while response was significantly improved when it was used as pollinator in the cross A188 x Lo904. In this specific case, the cross of Lo904 with B79 also gave favorable results in culture. Although callus cultures were more hard and compact than in the cross with A188, they were nevertheless more lasting and showed higher regenerative capability.
Friable, healthy growing calli at the third subculture of some of the crosses were then challenged as suspension culture. Finely dispersed suspensions were stably obtained in the following crosses: Lo1054 x A188, Lo904 x A188, Lo951 x A188 and their reciprocals. Interestingly, suspensions were also finely established from Lo904 in cross with A188; an unexpected result because of the very poor responsiveness of the callus culture.
The efficiency of plant regeneration was determined in each genotype.
Calli samples of about 1g fresh weight tissue, in triplicate, were fragmented
onto MS hormone-free-medium, in the light, and subcultured every 7 days
in order to stimulate maximal plant regeneration. In this respect also
the various genotypes differed consistently from each other and were grouped
in classes of regenerability depending on the number of regenerated complete
plantlets per gram fresh weight tissue. The best responding genotypes were
Lo964, Lo950, Lo951, Lo1056, and Lo1059 in combination with A188, and Lo881,
Lo1056, and Lo1059 in combination with B79. Their regenerative efficiency
paralleled, or even exceeded, the regenerative capability of the cross
between the two best responding genotypes A188 x B79. Suspension cultures
derived from the three genotypes mentioned above are currently being utilized
for protoplast isolation in order to establish an efficient regenerable
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