Simplified Regeneration Protocol for Cycas revoluta Thunb. Mature Zygotic Embryos

Mature zygotic embryos of Cycas revoluta Thunb. were used as explants to investigate direct and indirect organogenesis. Explants were incubated on half-strength Murashige and Skoog (½ MS) basal medium supplemented with various plant growth regulators, singly or in combination (all at 0.5 mg l-1): 6-benzyladenine (BA), kinetin (Kin), 2,4-dichlorophenoxyacetic acid (2,4-D), Kin×2,4-D, BA×Kin and BA×2,4-D. Cultures were placed at a low light intensity (4 µmol m-2 s-1 PPFD). Adventitious shoot regeneration was observed in the presence of 0.5 mg l-1BA after 35 days. The highest number of direct and indirect shoots per zygotic embryo was 3.67 and 29.67, respectively. Roots were induced on indirect shoots by continuous culture on rooting medium (½ MS,‏ 0.1 mg l-1 1-naphthaleneacetic acid) and hardened successfully in perlite. Each rooted plantlet with pinnate leaves and a primary tap root was individually isolated and acclimatized 185 days after the beginning of culture, with a 10% success rate.

Vegetatively reproductive red clovers (Trifolium pratense L.): An overview

Although the persistency of red clover (Trifolium pratense L.) can be a problem when grazed in mixed and pure stands, it is still regarded as a valuable source of high quality summer feed in some farming systems. Vegetatively reproductive red clover selections offer promise to solve this persistency problem but their growth, perennation, reproductive ability and productivity have not been investigated in New Zealand farming systems. This paper is an overview of a 4 year research programme involving nine experiments investigating these areas. The vegetatively reproductive red clovers evaluated were Astred, F2419 and Gualdo. There was no difference between Astred and Grasslands Pawera, a crown type red clover, in the total herbage accumulated over the first growing season under 4, 6 and 8 week grazing frequencies and lax and hard grazing intensities. After 3 years of grazing, significantly more Astred parent plants were alive compared to Pawera when rooted plantlets were counted as parent plants after one year. Varying numbers (0-66 plantlets/m2/yr) of rooted plantlets were produced by Astred in mixed and pure swards. More rooted plantlets were established per parent plant under wet surface soil conditions. Astred produced 57% of its autumn rooted plantlets on primary stems developed in September, or branches off these stems. Comparisons of the growth and morphology of Astred, F2419 and Gualdo are presented. Red clover selections that are vegetatively reproductive offer benefits to New Zealand farming systems and could solve some of the persistency problems currently experienced with red clover. Keywords: Astred, F2419, Gualdo, rooted plantlet, Trifolium pratense, vegetatively reproductive

REGENERATION AND AGROBACTERIUM TUMEFACIENS - MEDIATED TRANSFORMATION OF CUCUMBER (CUCUMIS SATIVUS L.).

We sought to develop efficient regeneratio nand transformation procedures for cucumber. Factors tested for regeneration included: hormone types and levels, genotype, explant source, and environmental conditions. Optimum regeneration was obtained using cotyledon pieces from 4 day old GY14A seedlings and culturing for 3 weeks under cool white lights (30-40 uE-2s-1) on MS medium supplemented with 1.0 mg/l 2,4-D, 0.5 mg/l BA, 0.3 mg/l ABA, 30 g/l sucrose, 1 g/l MES, and 3.07 g/l Scott gelrite. Shoots developed via somatic embryogenesis ca. 2 wk after explants were transferred to MS supplemented with 20 g/l sucrose, 1 g/l MES, and 4.37 g/l gelrite. Ca. 80% of the explants produce shoots, 1/3-1/2 of which produce rooted plantlets; total time from explant to rooted plantlet is ca. 8 wks. Transformation experiments utilized Agrobacterium tumefaciens strains LBA4404 bearing the CIBA-GEIGY pCIB10 vector with a selectable marker gene for kanamycin resistance. Optimal conditions include 45 mg/l kan, 10 min inoculation and 3 day co-cultivation. Preliminary evidence suggests that tobacco nurse cultures increase transformation efficiency. Transgenic plants were confirmed by Southern or dot blot analysis.

REGENERATION AND AGROBACTERIUM TUMEFACIENS - MEDIATED TRANSFORMATION OF CUCUMBER (CUCUMIS SATIVUS L.).

We sought to develop efficient regeneratio nand transformation procedures for cucumber. Factors tested for regeneration included: hormone types and levels, genotype, explant source, and environmental conditions. Optimum regeneration was obtained using cotyledon pieces from 4 day old GY14A seedlings and culturing for 3 weeks under cool white lights (30-40 uE-2s-1) on MS medium supplemented with 1.0 mg/l 2,4-D, 0.5 mg/l BA, 0.3 mg/l ABA, 30 g/l sucrose, 1 g/l MES, and 3.07 g/l Scott gelrite. Shoots developed via somatic embryogenesis ca. 2 wk after explants were transferred to MS supplemented with 20 g/l sucrose, 1 g/l MES, and 4.37 g/l gelrite. Ca. 80% of the explants produce shoots, 1/3-1/2 of which produce rooted plantlets; total time from explant to rooted plantlet is ca. 8 wks. Transformation experiments utilized Agrobacterium tumefaciens strains LBA4404 bearing the CIBA-GEIGY pCIB10 vector with a selectable marker gene for kanamycin resistance. Optimal conditions include 45 mg/l kan, 10 min inoculation and 3 day co-cultivation. Preliminary evidence suggests that tobacco nurse cultures increase transformation efficiency. Transgenic plants were confirmed by Southern or dot blot analysis.