Induction of Embryogenic Microspore in Oil Palm (<i>Elaeis guineensis Jacq</i>) by Starvation and Temperature Stress

Abstract

Gametophytic development (in vivo) of microspore was programmed to develop as pollen. By in vitro culture technique, normal development of microspores could be converted into embryogenic microspores. Developmental stages and stress were become the main roles of inducing the gametophytic pathway to embryo development. Embryogenic microspores then could be regenerated as haploid plants. Haploid culture technology can be used as a great solution to produce pure line parental with double haploid plants efficiently. The aim of this research was to determine the developmental stage of oil palm microspores and to induce normal development of oil palm microspores into embryogenic microspores by pre-treatment stress, i.e. carbohydrate starvation and temperature stress.

Oil palm microspores were isolated from fresh spikelet (from the tip, middle and basal part vertically of spikes) and then were observed in the samples stained with DAPI (4,6-diamidino-2-phenylindole) under electron microscopes to determine nucleus position. Late uninucleate microspores were then cultured aseptically in the carbohydrate-starvation medium and each was incubated to 4ºC, 25ºC and 34ºC for 2, 4 and 6 days as treatments. Further treatment was sub-culturing of embryogenic microspores into A2 embryogenesis medium and then embryogenic microspores were incubated to25ºC. Observation of embryogenic microspores and symmetrical divisions were determined in the samples stained with DAPI (cytology observation) while viability observation was carried out in the samples stained using FDA (fluorescence diacetate). Percentage data of viable embryogenic-microspores with symmetrical division and multicelluler structures were collected as representative of quantitative data.

The results showed that basal part of spike contains the most mature stage of microspore development and Marihat was become the highest proportion contained late uninucleate stage of microspore with 71.26% at the tip position of spike and followed by 55.94% at the basal spike. That was characterized by brown to dark-brown color of morphological spike. Pre-treatment with 4º Ctowards spikeletwas able to retain the viability of microspore(59,7% from total population) until 6 days and decline until 35,9% at 12 days after treatment. Pre-treatment temperature and starvation stress were able to induce embryogenic microspore which is characterized by swollen cell with oval structure, cytoplasm of cell was fulfilled by smooth granules. Embryogenic microspore developed to symmetrical division (2,1%) and multinucleat structures (4 nuclei) in A2 embryogenesis medium. Symmetrical division and multinucleat structures were become thefirst step in producing haploid plants wherein subsequently continue to double chromosome technique for double haploid plants production (pure line).