DISCUSSION

Plant growth regulator treatment, source of explant (greenhouse vs. in vitro), plant genotype, and time significantly influenced the number of explants producing calli. The reduced number of explants producing calli with increasing PGR concentration may be due to the phytotoxic effect developing at higher concentrations. 2,4- dichlorophenoxyacetic acid, a potent auxin, at high concentrations is herbicidal and at lower concentrations would inhibit plant growth. Phytotoxicity was evident in the appearance of explants grown on CI media containing high 2,4-D concentrations where explants became chlorotic and died. Another factor contributing to the chlorotic condition of explants is the length of time they were removed from the mother plant. However, this time effect is expected to be minimal as explants were placed on growth media containing nutrients and a carbohydrate source to promote growth and development. In addition, explants grown on media without PGRs remained green.

Although calli were produced on explants at most PGR treatments, only treatments 0.3,

1 .0, 3.0 and 10 flM 2,4-D and a combination of 1 .0 flM NAAJ 0. 1 flM Kinetin produced calli for all cultivars. More importantly, these five PGR treatments produced embryogenic calli that could be used in further studies on somatic embryo development.

Several factors influenced the initiation of embryogenic calli in asparagus. Genotype, PGR type and concentration are known to influence both the frequency of embryogenic calli development from explants and the development of asparagus somatic embryos from calli (Ghosh & Sen 1992; May & Sink 1996). The present results are consistent with these reports. Further, it is shown that the growth environment of the mother plant also influences the frequency of callus development on explants. Additional factors, such as developmental and physiological stage of development of explants, plus the growth environment of explant source, can also influence the efficiency of embryogenic calli development (Litz & Conover 1982; Monmarson et al. 1995). These factors could explain some of the results obtained in this study, in particular the difference in performance of explants collected from different sources.

3.1.5 SUMMARY

Plant genotype, PGR type and concentration, and length of time in culture influenced both the initiation of calli and the type of callus produced on explants. High PGR concentrations inhibited callus development. Concentrations of 0.3, 1 .0, 3.0, and 10 11M and 1 .0 11M NAN 0. 1 11M Kinetin produced embryogenic calli in each cultivar.

Asparagus somatic embryogenesis: 2. Somatic embryo development, maturation and regeneration

Abstract

Several somatic embryo induction, maturation and regeneration protocols were investigated for their effectiveness to promote embryo development from embryogenic calli developed from three different asparagus genotypes. The three cultivars used in this experiment were Aspiring (ASP), Karapiro (KP) and Pacifica (PC). The following sequence was found to be most effective in producing complete plantlets from embryogenic calli: callus induction (CI) on Murashige and Skoog (MS) media containing 3% sucrose, 1 % agar and either of 1 .0, 3.0 and' 10 � 2,4-D, followed by transfer onto liquid embryo induction media (EI) containing MS + 6% sucrose and finally regeneration on regeneration media (Rg4) containing MS + 0.2 gil glutamine + 3% sucrose + 1 % agar. Globular embryos were produced by all cultivars; however, only globular embryos developed from cultivar PC converted to plantlets. Treatment of 'PC' globular embryos at - 1 SoC (due to equipment failure) produced the highest percent converted plantlets (34 and 26% for 6-month-old embryogenic calli and 1 year-old embryogenic suspension cells respectively). Precocious germination, rhizogenesis, recurrent embryogenesis and organogenesis were common problems encountered. Further studies are required to understand the molecular and biochemical events involved in triggering and switching off embryogenic gene expression.

Keywords

Asparagus officinalis,

abscisic acid (ABA), osmotic potential, polyethylene glycol (PEG), embryo conversion, recurrent embryogenesis, rhizogenesis, organogenesis, precocious germination

3.2 EXPERIMENT 2

3.2.1 INTRODUCTION

Successful morphogenesis of somatic embryos from the globular embryo stage to

complete plantlet is dependent on several factors including polar auxin transport (West &

Harada 1 993; Zimmerman 1 993), plant species, genotype, age of plant cells (Attree et al.

1 990a; Karunaratne et al. 1 99 1 ), and the type and concentration of plant growth regulators

used to initiate and maintain embryogenesis (Borkird et al. 1 986; Smith & Krikorian

1 990). Related research on asparagus somatic embryogenesis shows different PGRs can effectively enhance development of somatic embryos, Li and W olyn ( 1 995) showing that

ancymidol, abscisic acid (ABA), uniconazole, and paclobutrazol could be used to enhance

development and conversion of bipolar embryos of 'G44T asparagus cultivar.

Carbohydrates included in the embryo induction media can influence asparagus embryo development as transfer of globular embryos from embryo induction media containing

high carbohydrate levels (4 - 10%) to media containing lower carbohydrate levels resulted

in a 2 to 4 times increase in conversion of asparagus somatic embryos (Levi & Sink

1 992). In an earlier study, Levi and Sink ( 1990) show that embryo conversion rates could

also be increased by transferring globular embryos grown on media containing 5% sucrose to media containing 5% fructose.

After development of bipolar somatic embryos a period of maturation and developmental arrest occurs (West & Harada ( 1 993). This period of maturation is an essential requirement for the accumulation of protein, carbohydrate and lipid reserves. Failure of somatic embryos to store these reserves may result in physiological disorders such as precocious germination, producing a large number of malformed embryos, and failure of embryos to convert to complete plantlets (Merkle et al. 1 995 ; Reuther 1 996). Abscisic acid and low osmotic potentials could play an important role in the accumulation of storage reserves required for somatic embryo maturation and conversion.

Chapter 3 Somatic embryo development, maturation and regeneration 42

There are no detailed reports on embryo maturation studies conducted on asparagus embryogenesis, and no reports documenting the types of physiologically abnormal embryo development. For asparagus somatic embryogenesis to be a viable alternative for the commercial propagation of asparagus clones, more research is required to explore the factors influencing embryo maturation and conversion.

This study focused on the development of protocols for the development, maturation and conversion of somatic embryos derived from embryogenic calli of three asparagus clones. Examples of physiologically abnormal somatic embryos produced during the study are described.

In document Asparagus somatic embryogenesis : detection of somaclonal variation using molecular and cytological analyses : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Horticultural Biotechnology at Massey Uni (Page 61-65)