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A reliable plant regeneration procedure by organogenesis has been obtained for Spinacia oleracea L. cv. Hybrid 102. The optimum procedure utilises a sequence of three different media. Explants from sterile seedling cotyledons or hypocotyls are incubated for 4 weeks in the dark on medium containing 4.6 μM kinetin and 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The calli are then transferred to medium without 2,4-D but containing kinetin and gibberellic acid (GA3). From this point on, incubation is in the light. A number of passages (usually three) of 4-6 weeks are required on this medium for shoot formation to occur. Calli destined to form shoots usually first undergo some root proliferation ('rooty calli'). Shoots can be induced to form roots by transfer to hormone-free medium. It is suggested that GA3 stimulates the development of shoot primordia induced by 2,4-D. The presence of kinetin (but not 6-benzylaminopurine) results in more rapid shoot formation.

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Medicago truncatula Gaertn. (barrel medic) is a pasture legume and a model plant for studying the molecular genetics of the Rhizobium-legume symbiosis. Using the highly regenerable seed line Jemalong 2HA and Agrobacterium tumefaciens (LBA4404), Medicago truncatula can be reliably transformed. The binary vector utilised was pBI121 containing the nptII and uidA genes under the control of the NOS and CaMV 35S promoters, respectively. Kanamycin-resistant embryogenic calli were produced from 17% of the initial leaf explants and on average each one of these embryogenic calli produced a single regenerated plantlet. Regenerated plants showed varying gene copy number and gene location. GUS activity was detected in roots, stems and leaves. The progeny of a regenerated plant containing a single gene copy segregated in a Mendelian fashion. This study confirms the value of Jemalong 2HA for producing transgenic plants for biological and agricultural studies.

A study has been made of factors that influence the yield, stability in culture, and ability to regenerate cell walls of isolated spinach (Spinacia olevacea L.) mesophyll protoplasts. The presence of 7 mM CaZ + or 7 mM Mg2+ in the isolation medium, which also included 1.5 % (w/v) Driselase, 0.25 % (w/v) pectinase and 0.8 M sorbitol, increased the yield and stability in culture of the protoplasts in a liquid nutrient medium. This latter medium has been used previously in the culture of spinach leaf discs, and does not support the division of spinach mesophyll cells. Protoplasts isolated in the presence of CaZ+ showed a greater capacity for cell wall regeneration compared with protoplasts isolated in the absence of ions or in the presence of Mg2+. Though darkness during culture improved the initial protoplast stability, it inhibited wall regeneration. The initial stability of protoplasts appeared to be dependent on plasma membrane stability, but after a few days in culture the most effective treatments were those which stimulated cell wall regeneration. In many cases, associated with cell wall regeneration, there was a budding off of small vesicles which sometimes contained chloroplasts. Protoplasts isolated in the presence of Ca2+ and incubated in low light had a 50% survival rate after 8 days culture and most had regenerated cell walls. Such culture of spinach protoplasts has not been shown previously and should be useful in developmental studies of spinach chloroplasts.